A Novel ARID DNA-Binding Protein Interacts With SymRK and Is Expressed During Early Nodule Development in Lotus Japonicus1[C][W][OA]

By Zhu, Hui Chen, Tao; Zhu, Maosheng; Fang, Qing; Kang, Heng; Hong, Zonglie; Zhang, Zhongming

During the establishment of symbiosis in legume roots, the rhizobial Nod factor signal is perceived by the host cells via receptor-like kinases, including SymRK. The NODULE INCEPTION (NIN) gene in Lotus japonicus is required for rhizobial entry into root cells and for nodule organogenesis.We describe here a novel DNA- binding protein from L. japonicus, referred to as SIP1, because it was identified as a SymRK-interacting protein. SIP1 contains a conserved AT-rich interaction domain (ARID) and represents a unique member of the ARID-containing proteins in plants. The C terminus of SIP1 was found to be responsible for its interaction with the kinase domain of SymRK and for homodimerization in the absence of DNA. SIP1 specifically binds to the promoter of LjNIN but not to that of LjCBP1 (a calcium-binding protein gene), both of which are known to be inducible by Nod factors. SIP1 recognizes two of the threeAT- rich domains present in the NIN gene promoter. Deletion of one of the AT-rich domains at the NIN promoter diminishes the binding of SIP1 to the NIN promoter. The protein is localized to the nuclei when expressed as a red fluorescence fusion protein in the onion (Allium cepa) epidermal cells. The SIP1 gene is expressed constitutively in the uninfected roots, and its expression levels are elevated after infection by Mesorhizobium loti. It is proposed that SIP1 may be required for the expression of NIN and involved in the initial communications between the rhizobia and the host root cells. Legume plants are capable of acquiring nitrogen from rhizobacteria maintained symbiotically in specialized root organ nodules that form through a complex developmental process involving an exchange of signals between the host root cells and the bacteria. At the beginning of nodule organogenesis, specific flavonoid metabolites released by the legume roots notify soil rhizobia that a suitable host is nearby. Inside the bacteria, the flavonoids are recognized by a receptor protein known as NodD. The binding of NodD to these flavonoids activates the protein and promotes the transcription of other nod genes involved in the synthesis and secretion of Nod factors, the rhizobial signaling molecules (Peck et al., 2006). The common feature of all Nod factors is the presence of a chitin backbone and a fatty acyl tail (Denarie et al., 1996). Rhizobial Nod factors, in turn, are capable of inducing a series of specific responses in the host root cells, including root hair deformation, alkalinization of the cytosol, depolarization of the plasma membrane, and calcium influx and spiking (Ehrhardt et al., 1992, 1996; Kurkdjian, 1995; Felle et al., 1995, 1996; Limpens and Bisseling, 2003; Gleason et al., 2006). Root hair deformation and curling are the earlymorphological changes induced by rhizobia. Purified Nod factors can cause root hair deformation at concentrations as low as 10212 M (Lerouge et al., 1990; Spaink et al., 1991; Sanjuan et al., 1992; Margaert et al., 1993), although in most cases curling is only observed when the bacteria are present (Relic et al., 1993).

The perception and signal transduction of Nod factors in the host cells have been subject to intense molecular and genetic studies in recent years. Rhizobial Nod factors have been shown to be recognized by lysin motif (LysM)-containing receptor-like kinases (RLKs), such as NFR1/NFR5 from Lotus japonicus and LYK3 and NFP from Medicago truncatula (Madsen et al., 2003; Radutoiu et al., 2003; Limpens et al., 2003; Arrighi et al., 2006). The LysM RLKs are localized to the plasma membrane with the LysM domain protruding to the extracellular space and the kinase domain facing the cytoplasm. The LysM domain interacts with the lipochitin-oligosaccharide backbone of Nod factors, resulting in Nod factor recognition (Steen et al., 2003, 2005; Radutoiu et al., 2007). Using forward genetics and map-based cloning approaches, a series of host genes involved in the perception of the Nod factor signals, including SymRK (symbiosis RLK), Castor, Pollux, Nup133, Nup85, CCaMK, and Cyclops, have been characterized from the legume L. japonicus (Kistner et al., 2005). Mutations in any of these genes result in defects in nodule initiation. Downstream of this pathway, putative transcription factors encoded by LjNIN, LjNSP1, and LjNSP2 (Schauser et al., 1999; Heckmann et al., 2006) are also required to initiate nodule organogenesis.

SymRK from L. japonicus encodes a protein with three Leu-rich repeat (LRR) domains in the predicted extracellular region. Mutations in the SymRK gene abolish the host interaction with rhizobia (Stracke et al., 2002). Its orthologs, NORK, DMI2, and SYM19, from Medicago sativa, M. truncatula, and Pisum sativum, respectively, have been cloned (Endre et al., 2002; Stracke et al., 2002). The LRR-RKs represent the largest group of receptor kinases in plants, comprising approximately one-half of the predicted receptor kinases in Arabidopsis (Arabidopsis thaliana; Shiu et al., 2004). LRR-RKs have been implicated in diverse plant signaling pathways, including the perception of pathogen signals, brassinosteroid hormones, and the CLAVATA peptide hormone (Oldroyd and Dowine, 2004). It has been proposed that the three LRRs in SymRK may be involved in protein-protein interactions, proteinligand interactions, or autophosphorylation-regulated kinase activation (Yoshida and Parniske, 2005). The observation that the symRK/nork mutant fails to form arbuscular mycorrhiza suggests that SymRK/NORK plays a role in the exchange of signals with both symbiotic bacteria and fungi. Although SymRK is known to be essential in the Nod signaling pathway in L. japonicus, the biochemical mechanism leading to Nod factor-induced transcriptional activation is obscure. Moreover, how the kinase activity of SymRK is regulated remains to be determined. Recently, a NORKinteracting partner in M. truncatula has been identified as 3-hydroxy-3-methylglutaryl-CoA reductase 1, which may link the Nod signaling pathway with the formation of isoprenoid-derived phytohormones (Kevei et al., 2007).

In this study, we demonstrate that a novel protein, designated as SIP1 for SymRK-interacting protein 1, interacts with the kinase domain of SymRK. SIP1 is a transcription factor containing an AT- rich interaction domain (ARID) and may participate in Nod factorinduced transcriptional activation of genes required for nodule initiation.

RESULTS

Characterization of a SymRK-Associated Protein

The SymRK peptide contains three LRRs, a transmembrane domain, and an intracellular kinase domain. The structure features suggest potential roles of SymRK in the perception of extracellular signals and transduction of the signals through the intracellular kinase domain (Stracke et al., 2002). In an attempt to identify SymRK- associated proteins, we used the kinase domain of SymRK as a bait of the yeast two-hybrid system and screened a Lotus cDNA library constructed in the prey vector pGADT7-Rec2. Approximately five million yeast Saccharomyces cerevisiae colonies expressing the cDNA library were assayed for their ability to grow on selective synthetic dextrose medium (SD/-Leu-Trp-His-Ade). The bait plasmids were isolated from positive colonies and reintroduced back to yeast cells containing the prey plasmid. Colonies that failed to grow in the second round of testing were considered as false positives. After eliminating false positives, several clones were identified as potential interaction partners of SymRKprotein kinase (PK). One positive cDNAwas isolated from three independent yeast colonies. It encoded a novel protein designated SIP1 (Fig. 1A; Supplemental Fig. S1).

The full-length SIP1 cDNA (GenBank accession no. EU559710) contained an open reading frame of 1,224 nucleotides encoding a peptide of 408 amino acid residues with a predicted molecular mass of 45.7 kD. Analysis of the peptide sequence revealed the presence of a conserved ARID (Fig. 1B) that has been implicated in sequence- specific DNA binding (Gregory et al., 1996). ARID-containing proteins appear to play important roles in diverse biological functions, including cell proliferation and differentiation, and organ development (Herrscher et al., 1995; Gregory et al., 1996; Kortschak et al., 2000). The mouse BRIGHT (B-cell regulator of immunoglobulin H transcription) protein and the fruit fly DRI (Drosophila dead ringer) are two well-characterized ARID-containing transcription factors (Herrscher et al., 1995; Gregory et al., 1996; Valentine et al., 1998; Iwahara et al., 2002; Wilsker et al., 2005). The predicted three-dimensional model of the ARID domain of SIP1 consists of eight a-helices, two b-strands, and four structure- undefined loops (Fig. 1B). The structural features of this 91- residue motif were identical to those found in animal ARIDcontaining proteins such as DRI (Fig. 1B; Iwahara and Clubb, 1999) and indicated that SIP1 might bind to the AT-rich region of plant promoters.

ARID-containing proteins are widely present in plant genomes. Ten such proteins have been found in Arabidopsis (Fig. 1C) and can be grouped into four subfamilies, designated high-mobility group (HMG), EGL-27 and MTA1 homology 2 (ELM2), plant homeodomain (PHD), and heat stress protein 20-like (Hsp20)-related proteins (Fig. 1D). Animal proteins containing HMG and ELM2 motifs have been implicated in DNA binding and transcription regulation (Ding et al., 2003; Stros et al., 2007). PHD-containing proteins are known to be involved in binding to methylated histone H3 (Ramon-Maiques et al., 2007). Hsp20- like proteins, also known as a-crystallin domain-containing proteins, are believed to function in protecting other proteins from denaturation by heat (Scharf et al., 2001). All of these proteins contain at least another motif in addition to ARID. However, there was no other recognizable motif except ARID in the Lotus SIP1. Phylogenetic tree analysis (Fig. 1D) revealed that SIP1 is closely related to the Hsp20 subfamily of ARID proteins in Arabidopsis. The Hsp20 subfamily is characterized by the presence of an Hsp20-like domain in the C terminus of the protein. The C terminus of SIP1 does not contain the Hsp20-like domain, but has instead evolved to perform a new function, i.e. interaction with SymRK (see below). Taken together, our data suggest that SIP1 may have evolved from the Hsp20 subfamily of ARID-containing proteins during evolution and acquired the ability to act as a transcriptional regulator involved in the Nod factor signaling in L. japonicus. Interaction between SymRK and SIP1

To determine which domain of SIP1 is responsible for its interaction with SymRK-PK, we constructed a series of SIP1 deletions in pGADT7 (Fig. 2A). The N-terminal half of SIP1 (SIP1N) containing the N terminus and the ARID domain was not found to interact with SymRK-PK. The ARID domain alone, SIP1A, also did not interact with SymRK-PK. However, yeast colonies expressing the C terminus of SIP1, SIP1C, were able to grow on the selection medium lacking His (SD/- Leu-Trp-His) and exhibited significant b-galactosidase activities (Fig. 2C), suggesting that the C-terminal 184 amino acid residues of SIP1 are critical for its interaction with SymRK. This interaction was further confirmed using an in vitro protein-protein interaction assay (Fig. 2D). For this assay, SIP1 and its deletion fragments were expressed as His-or chitinbinding domain (CBD)-tagged recombinant proteins and immobilized to nickel beads or chitin beads. After incubation of the beads with purified SymRK-PK, followed by washing with buffer, proteins retained to the beads were eluted in SDS sample buffer and resolved on SDS-PAGE. The presence of SymRK on the beads was detected by immunoblotting with the anti- SymRK antibody. As shown in Figure 2D, only the full-length SIP1 and SIP1C could pull down SymRKPK, confirming that the C terminus of SIP1 was responsible for its interaction with SymRK.

SIP1 Binds AT-Rich Double-Stranded DNA

The ARID motif of SIP1 contains a noncanonical helix-turn-helix motif (helices H5 and H6) for potential DNA binding. To test this DNA-binding activity, we purified His-tagged SIP1 and performed electrophoretic mobility shift assays (EMSA) with a ^sup 32^P- endlabeled double-stranded DNA trimer of NP^sub 3^ or TTA^sub 9^, the consensus binding sites of the Drosophila homeodomain protein Engrailed (Gregory et al., 1996). The results showed that SIP1 bound both NP^sub 3^ and TTA^sub 9^ (Fig. 3A) and could be competed out by 10-fold excess of unlabeled NP^sub 3^ oligonucleotide trimer (Fig. 3A, lane 5).

We further examined whether SIP1 contains a transcription activation domain (AD). We constructed plasmids that would express the GAL4 DNA-binding domain fused with SIP1 or its deletion fragments. The plasmids were transferred into the yeast strain AH109 that expressed ADE2, HIS3, lacZ, and MEL1 reporter constructs under GAL4-responsive promoters (CLONTECH). If SIP1 contained an AD, the SIP1 fusion protein should bind to the GAL4-responsive promoters and drive the expression of the ADE2, HIS3, lacZ, and MEL1 reporters. A known transcription activator, NSP1 (Smit et al., 2005; Heckmann et al., 2006), was used as a positive control and proved able to allow yeast colonies to grow on SD/-Trp-His and SD/-Trp-Ade media (Fig. 3B). However, as shown in Figure 3B, colonies expressing SIP1 and its deletion fragments could not grow on these media, indicating that SIP1 did not contain a transcription AD (Fig. 3B). It would be interesting to test if SIP1 forms heterooligomers with other proteins that contain an activator or repressor domain.

SIP1 Binds Specifically to the NIN Promoter

The expression of the NIN (nodule inception) and CBP1 (calcium- binding protein 1) genes is induced by Nod factors (Schauser et al., 1999; Webb et al., 2000; Radutoiu et al., 2003). To test if SIP1 plays a role in the induction of NIN and CBP1 by Nod factors,we cloned the promoter regions of both genes and assayed for their affinity with SIP1 in the yeast one-hybrid system. The 500-bp NIN promoter (Borisov et al., 2003) and the 482-bp CBP1 promoter (Webb et al., 2000) were inserted in front of the HIS3 reporter, generating NIN^sub pro^::HIS3 and CBP1^sub pro^::HIS3 constructs. SIP1 was expressed as a fusion protein with the GAL4 AD. This plasmid (pGAD-SIP1), along with one of the reporter constructs, was cotransformed into yeast Y187 cells. If SIP1 could bind to the promoter, the GAL4 AD would drive the expression of the HIS3 reporter, and the yeast cells would grow on SD/-Trp-Leu-His plates supplemented with 3-amino-1,2,4-triazole (3-AT). The results showed that the cotransformants of pGADSIP1 and NIN^sub pro^::HIS3 (Fig. 4C, colony 3) were indeed able to grow on the selective plates, whereas the cotransformants of pGAD-SIP1 and CBP1^sub pro^::HIS3 (Fig. 4C, colony 5) did not grow, indicating that SIP1 specifically binds to the NIN promoter but not the CBP1 promoter.

A search of the promoter region (about 4 kb upstream of the first ATG) of the NIN gene identified three potential AT-rich motifs, located in the regions from -2,299 to -2,287, from -393 to -367, and from -69 to -59 bp, respectively (Borisov et al., 2003; Fig. 4A). To determine whether SIP1 binds to these sequences in vitro, we synthesized three oligonucleotides corresponding to Oligo1 to Oligo3 (Fig. 4A) and performed EMSA assays using purified SIP1. As shown in Figure 4B, SIP1 specifically recognized and bound to Oligo1 and Oligo3 but not Oligo2.

Because the 500-bp NIN promoter fragment used in the yeast one- hybrid assay (Fig. 4A) did not contain Oligo1 (Fig. 4C), we hypothesized that Oligo3 should be the AT-rich site for SIP1 binding. We further reasoned that deletion of the Oligo3 site from the NIN promoter should diminish the ability of the yeast cells to grow on SD-His13-AT medium. We removed a 70-bp fragment (-1 to -69 bp) from the NIN promoter and showed that the DeltaNIN promoter lost the binding site for SIP1 and yeast cells were no longer able to grow on the selection medium (Fig. 4C, colony 4). Taken together, our data demonstrate that SIP1 specifically recognizes the Oligo3 AT- rich site of the NIN promoter and may be required for the Nod factorinduced NIN gene expression.

SIP1 Dimerization

The mouse BRIGHT protein forms a tetramer that binds to DNA (Herrscher et al., 1995) but exists as a dimer in the absence of DNA (Nixon et al., 2004). To determine whether SIP1 dimerizes in the absence of DNA, we coexpressed two SIP1 fusion proteins, one with the GAL4 DNA-binding domain (SIP1-BD) and the other with the GAL4 AD (SIP1-AD) in the yeast two-hybrid system. The N and C termini and the ARID domain of SIP1 were also fused with the GAL4 AD, generating SIP1N-AD, SIP1C-AD, and SIP1A-AD. As shown in Figure 5A, full- length SIP1-AD interacted with SIP1-BD, suggesting that SIP1 dimerizes. The interactions were also detected when the C terminus of SIP1 was used instead (SIP1C with SIP1; SIP1C with SIP1C; Fig. 5A), suggesting that the C-terminal 184 residues are responsible for SIP1 dimerization in the absence of DNA. In contrast, the N terminus (223 residues) of SIP1, which contains the ARID domain, was not required for SIP1 dimerization.

To further confirm this interaction, we immobilized glutathione S- transferase (GST)-SIP1, CBD-SIP1N, and CBD-SIP1C fusion proteins on GST beads or chitin beads and incubated the beads with His-tagged SIP1, SIP1N, and SIP1C, respectively. After washing, the proteins retained on the beads were subjected to immunoblotting with the anti- His-tag antibody. As shown in Figure 5B, GST-SIP1 and CBD-SIP1C could pull down SIP1 (lanes 1 and 3), whereas CBD-SIP1N could not (lane 2). Moreover, CBD-SIP1C was able to pull down SIP1C (lane 6), suggesting the C termini are sufficient to form dimers.

Induction of SIP1 Gene Expression by Rhizobial Infection

SymRK is expressed constitutively in the roots of Lotus, and its mRNA level does not change upon the treatment with Nod factors for 24 and 48 h or after the inoculation with Mesorhizobium loti (Stracke et al., 2002). The NIN gene, on the other hand, is not expressed in the uninfected roots and is induced by treatments with Nod factors or rhizobial infection (Schauser et al., 1999; Radutoiu et al., 2003). Using quantitative PCR, we examined the SIP1 and NIN mRNA levels in different tissues of L. japonicus. SIP1 mRNA was expressed constitutively in leaves and control (uninoculated) roots (Fig. 6A). In the young roots (2 d old), SIP1 expression levels were low but increased to a steady state after 6 to 8 d. In the stem, its expression levels were relatively low but detectable. In contrast, NIN expression levels were relatively low in stems, leaves, and control roots (Fig. 6B).

We then focused on the expression levels of SIP1 and NIN in roots after infection with M. loti. When inoculated with M. loti, an induction of SIP1 mRNA was observed as early as 5 h post inoculation (hpi). In 24 hpi, the expression levels dropped down to a steady level, which was slightly higher than that observed in the control roots. This expression pattern was distinct from that of NIN (Fig. 6B), which exhibited significant induction 5 h after rhizobial inoculation and maintained a high expression level in inoculated roots. It is important to note that the timing of the SIP1 induction (5 hpi) correlates well with that of NIN. After the initial induction, NIN continued to be expressed at relatively high levels. In conclusion, the SIP1 gene is expressed constitutively in roots and leaves, and its expression levels are elevated in roots transiently (5 hpi-1 d postinoculation [dpi]) after rhizobial infection. It may play a role in the induction of the NIN gene during the process of rhizobial entry and nodule organogenesis. Subcellular Localization of SIP1

To determine the subcellular localization of SIP1, we expressed SIP1 as a fusion protein with the Discosoma red fluorescent protein (DsRed) under the control of the cauliflower mosaic virus 35S promoter. The fusion protein was transiently expressed in the onion (Allium cepa) epidermal cells via particle bombardment, and its expression was monitored using a confocal laserscanning microscope. As expected in the control cells expressing DsRed alone, the red fluorescence was detected only in the cytoplasm (Fig. 7, D-F). In the onion epidermal cells expressing the SIP1-DsRed fusion protein, the red fluorescence was concentrated to the nuclei (Fig. 7, A-C). This result is consistent with its potential function in DNA binding and transcription regulation. However, it remains to be determined how SIP1 interacts with SymRK in Lotus roots after rhizobial infection.

DISCUSSION

SymRK is a member of a large family of LRR RLKs in plants and is required for the legume root cells to perceive the Nod factor signals released by the infecting rhizobia (Yoshida and Parniske, 2005). NIN is a key transcription factor required for rhizobial entry into the root cells and for nodule organogenesis (Schauser et al., 1999; Radutoiu et al., 2003). In this report, we describe a novel DNA-binding protein, SIP1, which may provide a link between SymRK and NIN. We demonstrate that SIP1 interacts with the intracellular kinase domain of SymRK (Fig. 2) and specifically binds to the Oligo3 AT-rich site of the NIN promoter (Fig. 4). Because SIP1 does not contain a transcription AD (Fig. 3), it may form hetero-oligomers with other unidentified proteins that contain an activator or repressor domain. Plu-1, a human ARID-containing protein, also lacks an AD but contains transcriptional repressor properties and forms hetero-oligomers with other transcription factors (Tan et al., 2003). The initial signal communications between the rhizobia and roots involve a number of different molecular exchanges culminating in calcium spiking in the host cells. SIP1, by linking the SymRK receptor kinase to the NIN transcription factor, may play a pivotal role for the successful establishment of rhizobia-legume symbiosis.

SIP1 represents a new member of the conserved ARID family of proteins in plants. The ARID domain was initially identified in the mouse BRIGHT protein (Herrscher et al., 1995) and Drosophila DRI (Gregory et al., 1996). ARID-containing proteins have now been shown to be present in all sequenced eukaryotic organisms, including mammals, insects, plants, nematodes, and yeast (Wilsker et al., 2002). Although ARID is an a-helix-based DNA-binding domain, the structures and functions of ARID-containing proteins are highly diverse (Wilsker et al., 2002). These proteins appear to play vital roles in the regulation of development and tissue-specific gene expression.

The ARID domains, especially their 3-D structures, are conserved between animals and plants (Fig. 1B). However, other parts of the molecules can be very different among ARID-containing proteins in an organism. In Arabidopsis, the 10 ARID-containing proteins vary in length from 319 residues in At3g13350 to 786 residues in At2g17410. They can be grouped into four subfamilies on the basis of their phylogenetic relationship and the presence of functional motifs (Fig. 1D). L. japonicus SIP1 is closely related to the Hsp20 subfamily in Arabidopsis, although it does not contain an Hsp20- like domain. The Hsp20 motif is found in the C termini of the Arabidopsis ARID proteins (Fig. 1C). In L. japonicus SIP1, the C terminus has evolved into a different domain that is responsible for interacting with SymRK (Fig. 2) and for the homodimerization (Fig. 5). SIP1 represents a new ARID-containing transcription factor in plants and is potentially involved in linking the Nod signal perception to the transcriptional regulation during nodule development in L. japonicus.

The ARID domain has not been found in Archea and eubacteria, but is widespread in protozoa, metazoans, green algae, fungi, plants, and animals. There are 15 ARID proteins in the human genome and two in S. cerevisiae (Wilsker et al., 2002, 2005). The four Arabidopsis ARID subfamilies (Fig. 1D) do not correlate closely with the seven subfamilies of the human ARID proteins (Wilsker et al., 2005). At3g43240 is a single member of the Arabidopsis subfamily that possesses an ARID and a PHD domain. In humans, the most closely related proteins are the four ARID proteins in the JARID1 subfamily, which contain two or more PHD domains each, in addition to several other functional motifs (Wilsker et al., 2005), and are also twice as large as At3g43240. The remaining three subfamilies of Arabidopsis ARID proteins are plant specific, because no known human ARID protein contains an Hsp20, HMG, or ELM2 domain (Wilsker et al., 2002, 2005). This diversity in peptide length and motif arrangements suggests that the specific ARID subfamilies have apparently evolved independently after the divergence of plant and animal lineages.

ARID proteins in higher eukaryotes are involved in differentiation and transcriptional regulation of gene expression (Wang et al., 2007). At least two human ARID proteins are known to be induced during tumorigenesis. Plu-1 (JARID1B) was identified as an upregulated gene product in human breast cancer and is not expressed in normal adult tissues except testis. RBP1L1 (ARID4B) was identified as a tumor antigen and is highly expressed in all cancer cell lines (Wilsker et al., 2002; Tan et al., 2003). Nodule initiation involves the de-differentiation of the root cortical cells into the meristematic cells and the activation of a series of nodule-specific genes. Our study shows that SIP1 is constitutively expressed in roots, and its expression is highly induced 5 h after rhizobial infection (Fig. 6). Although L. japonicus SIP1 is not homologous to the human Plu-1 and RBP1L1 except within the ARID domain, they share some similarity in their function in reprogramming the differentiated cells back to the dividing status, i.e. tumorigenesis in human and nodule organogenesis in the legume.

There are three AT-rich motifs in the promoter of the NIN gene, two of which showed affinity with SIP1 in in vitro assays (Fig. 4). In the yeast one-hybrid system, the 500-bp promoter region of the NIN gene was found to contain cis-DNA elements for SIP1 binding. The binding of SIP1 to the NIN promoter was apparently brought about via the Oligo3 AT-rich motif, which is localized to the promoter region between -59 to -69 bp. Because SIP1 does not have a transcription AD (Fig. 3B), it is likely that SIP1 may function as a DNA sequence- specific binding protein and may form an oligomer with another transcription activator or repressor. We are currently in the process of searching for these potential SIP1-interacting partners and examining the biological functions of SIP1 and its associated proteins in nodule organogenesis in L. japonicus. Further analyses of the signaling pathway involving SIP1 may eventually lead to the identification of new molecular events required for the later steps in the symbiotic establishment in legume roots.

MATERIALS AND METHODS

Plant Materials

Seeds of Lotus japonicus MG-20 were surface-sterilized in 2% sodium hypochlorite for 8 min and washed seven times with sterilewater. The seedswere left to germinate for 48 h at 22[degrees]C on sterile water-soaked filter paper in petri dishes in a dark room. Seedlings were subsequently planted in pots on sterile sands supplemented with nitrogen-free Fahraeus medium (Fahraeus, 1957) and were grown in a growth chamber maintained at 22[degrees]C with a 16-h-light/8-h-dark cycle. Five-day-old seedlings were inoculated with approximately 107 CFU/mL of Mesorhizobium loti NZP2037. Roots were collected from plants 2, 4, 6, 8, and 12 d after rhizobial inoculation and were frozen in liquid nitrogen.

cDNA Library Construction

Total RNA was isolated from the equally mixed roots collected 2, 4, 6, 8, and 12 d after rhizobial inoculation using the TRIzol reagent (Invitrogen). Total RNA (2 mg) was reverse transcribed into single-stranded cDNA using oligo(dT) as the primer. The cDNA was size-fractionated using BD Matchmaker library construction and screening kits (BD Biosciences CLONTECH). The cDNA fragments longer than 500 bp were cotransformed with linearized pGADT7-Rec into yeast (Saccharomyces cerevisiae) AH109 cells. The transformants were selected on SD/-Leu medium according to the manufacturer’s instruction. The transformation efficiency was approximately 2 3 106 CFU/3 mg pGADT7-Rec.

Library Screening

The SymRK-PK cDNA (GenBank accession no. AF492655) was amplified by reverse transcription-PCR with the following gene-specific primers: 5′-GGGCATATGATGGAGAGGTACAAAACCTTG-3′ and 5′- AAAGAATTCTCTCGGCTGTGGGTGAG-3′. PCR was carried out using ExTaq DNA polymerase (TaKaRa) with an initial denaturation step of 95[degrees]C for 5 min, followed by 30 cycles of 95[degrees]C for 15 s, 55[degrees]C for 30 s, and 72[degrees]C for 1 min. After the last cycle, an extension step for 10 min at 72[degrees]C was carried out. Purified PCR products were inserted into the Nde1-EcoR1 site of pGBKT7 vector. Screening of interaction clones was carried out according to the manufacturer’s instructions (CLONTECH). A total of 1 3 107 transformants from the cDNA library were screened for growth on the SD/-Leu-Trp-His-Ade media. Positive clones were verified by retransformation of the rescued plasmid into the AH109 containing the bait plasmid (pGBKT7-SymRK-PK). Plasmid pGBKT7-Lam (CLONTECH) was used as a negative control. Colonies growing on the SD/-Leu-Trp- His-Ade media were transferred to selective media-containing X-Gal (80 mg/mL) or were left on filters as described (Ma et al., 2007). The blue colonies were characterized. b-Galactosidase Assay

Yeast cells grown in liquid selection mediawere pelleted and washed twice with Z-buffer (60 mM Na2HPO4, 40mM NaH2PO2, 10mM KCl, 1.0 mM MgSO2, pH 7.0). The cells were resuspended in 300 mL of Z- buffer and permeabilized by two freeze-thaw cycles in liquid nitrogen and 37[degrees]C. Cell extracts were added 0.7 mL of Z- buffer containing 50 mM b-mercaptoethanol and 160 mL of O- nitrophenyl b-D-galactopyranoside (4mg/mL) in Z-buffer. After incubation at 30[degrees]C for 30 min or until the yellow color appeared, the reaction was terminated by the addition of 0.4 mL of 1.0 M Na2CO3. The reaction mixture was centrifuged for 10 min at 14,000 rpm to remove cell debris. b-Galactosidase activity in the supernatant was measured at OD420 and expressed in Miller units (Miller, 1972).

Expression and Purification of Fusion Proteins

The full-length cDNA of SIP1 was inserted at the Nde1-Sal1 site of pET28 vector (Novagen), generating pET-SIP1. The 3′ region of SIP1 was amplified by PCR using primers 5′- CTTTCTAGACCATATGAAGTCCAGAGAAAGGA-3′ and 5′- TTTTGTCGACAAGCTTCACGGCTCCCTG-3′. The fragment was cloned into the Nde1-Sal1 site of pET28a vector, generating pET-SIP1C. The 5′ region of SIP1 was amplified using primers 5′-CCGGTACCCATATGGAAATTGTTGAT- 3′ and 5′-GGGAAGCTTGTCGACTTAGAGCTGTAACTC-3′. The fragment was cloned into the Nde1-Sal1 site of pTYB12 (New England Biolabs), yielding pTYB-SIP1N. For protein expression, Escherichia coli BL21 (Novagen) or ER2566 cells (New England Biolabs) harboring the plasmid were induced with 0.5 mM isopropyl 1-thio-beta-D-galactopyranoside in Luria-Bertani broth for 8 h at 16[degrees]C. His-tagged proteins were purified using nickel-agarose beads (Qiagen) according to the manufacturer’s instruction. CBD-tagged proteins were purified using chitin beads (New England Biolabs) as described previously (Zhang et al., 2003). Purified proteins were desalted by dialysis in PBS buffer and concentrated with PEG-8000 powder.

In Vitro Protein-Protein Interaction

To assay the interaction between SymRK-PK and SIP1, His-tagged SIP1 and SIP1C were bound to nickel-agarose beads, while CBD-SIP1N was absorbed on the chitin beads. The beads were incubated with 5 [mu]g of purified SymRK-PK protein in the interaction buffer (20 mM Tris-HCl, 100 mM KCl, 2mM MgCl2, 5% glycerol, pH 8.0) for 1 h on ice with gentle shaking. The nickel beads were washed three times with a buffer solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2mM KH2PO4, 100 mM imidazole, 5% glycerol, pH 8.0), whereas the chitin beads were washed three times with 1.0 mL of TEG buffer (20mM Tris-HCl, 1mM EDTA, 5% glycerol, pH 8.0). The retained proteins were eluted by boiling for 3 min in 1 x SDS sample buffer (2% SDS, 29.1 mmol/L Tris, pH 6.8, 10% glycerol, and 0.01% bromphenol blue; Zhang et al., 2003). Samples were analyzed on 10% SDS-PAGE, followed by immunoblotting with the anti-LjSymRK antibody.

To assay SIP1 dimerization, GST-tagged SIP1 was expressed and purified on glutathione-Sepharose 4B (Sigma) as described elsewhere (Zhang et al., 2000). CBD-tagged SIP1N and SIP1C were immobilized on chitin beads. The beads were incubated with purified His-SIP1, His- SIP1N, and His-SIP1C proteins for 1 h on ice with gentle shaking. Following washing, the retained proteins were resolved on 10% PAGE and detected using the anti-His-tag antibody.

EMSA

EMSA of SIP1 was performed on 5% nondenaturing polyacrylamide gels as described previously (Gregory et al., 1996). DNA-binding activity of SIP1 was examined using g-^sup 32^P-end-labeled double- stranded oligonucleotide trimer of the consensus Engrailed binding site NP^sub 3^ [(TCAATTAAATGA)X3] or trimer of TTA^sub 9^ [(TTATTATTA)X3; Gregory et al., 1996]. To test if SIP1 specifically binds to the promoter of the NIN gene, Oligo1, Oligo2, and Oligo3 (see Fig. 4A for sequences) were synthesized and used for EMSA. His- tagged SIP1 was purified with nickel-agarose beads and eluted using 500 mM imidazole followed by dialysis. Approximately 10 ng of SIP1 protein was added in a mixture containing 0.3 [mu]M of gamma-^sup 32^P-labeled double-stranded DNA in 10 vM Tris-HCl, pH 7.5, 50 mM NaCl, 1.0 mM EDTA, 1.0 mM dithiothreitol, 50 [mu]g bovine serum albumin, and 5% glycerol. After incubation at room temperature for 20min, the samples were subjected to electrophoresis on a 5% polyacrylamide/bis (29:1) gel in 0.5 x Tris-borate/EDTA buffer at a constant voltage of 100 V. The gel was dried, and the radioactive bands were detected using PhosphorImager (FUJIFILM).

Transient Expression

The SIP1 cDNA amplified using primers 5′- TATACTAGTATGGAAATTGTTGAT-3′, and 5′-AAGGTCACCTTACACGGCTCCCTG-3′ was cloned into the Spe1/BstEII site of pCAMBIA1302 vector (CAMBIA). We amplified DsRed coding sequence using primer 5′- CAACTAGTATGAGGTCTTCCAAGAATGT-3′, and 5′- TTACTAGTAAGGAACAGATGGTGGCGTC-3′ from the pDsRed (CLONTECH), and it was inserted into the Spe1 site of the pCAMBIA1302-SIP1 construct, resulting in p35S-DsRed-SIP1. The plasmid was used for transient expression in the onion (Allium cepa) epidermal cells by particles bombardment using a Biolistic PDS-1000/He particle delivery system (Bio-Rad). After incubation for 24 to 48 h at 25[degrees]C in dark, the epidermal cell layers were examined using a confocal laser- scanning microscope (Leica).

Yeast One-Hybrid Assay

The SIP1 cDNAwas fused to the GAL4 AD in pGADT7-Rec2 (CLONTECH), generating pGADT7-SIP1. The promoter (-1 bp to -500 bp) of the NIN gene (Schauser et al., 1999) was amplified using primers 5′- GGAATTCGGCGTGAGACTTTACATTAC-3′ and 5′-CGACGCGTGAAAATGATTGCCTTGCC- 3′. A 430-bp fragment of the NIN promoter (-70 to -500 bp) lacking the Oligo3 AT-rich site (DeltaNIN) was amplified by PCR using primers 5′-GGAATTCGGCGTGAGACTTTACATTAC-3′ and 5′- TTTTACGCGTCTTGCTAGGACC-3′. The promoter of the LjCBP1 gene (Webb et al., 2000) was amplified from genomic DNA using primers 5′- GGAATTCCCAAAGTCAGTCAATTTTTG-3′ and 5′-CGACGCGTCCACTGACTTAATTCATG- 3′. The promoter fragments of the NIN, DeltaNIN, and LjCBP1 genes were inserted into the EcoRI-MluI site of pHIS2 (CLONTECH), producing NIN^sub pro^::HIS3, DeltaNINproT HIS3, and CBP1^sub pro^::HIS3, respectively, which would express the HIS3 reporter under the control of the corresponding promoter.

The plasmid was transformed into yeast Y187 cells harboring pGADT7-SIP1, which expresses SIP1 fused with the GAL4 transcription AD. The DNA-binding activity of SIP1 was determined by the expression of the HIS3 reporter.

Measurement of Transcript Level by Quantitative PCR

Total RNA was isolated from stems, leaves, control roots, and roots inoculated with M. loti, using a TRIZOL reagent (Invitrogen). RNA samples were treated with DNase I to remove potential contaminating genomic DNA, followed by extraction with phenol:chloroform. The DNA-free RNA samples were confirmed by lack of a PCR product using primers specific to the untranscribed NIN gene promoter (5′-GTTTTCAAGAATGGGAGGGG-3′, 5′-CTCCTCTGGTTTCATTGGTG- 3′). First-strand cDNA was prepared using Oligo(dT) primer, and quantitative PCR was performed on a Mini Opticon real-time PCR system (Bio-Rad) using One-Step SYBR PrimeScript RT-PCR kit II (Takara). The same cDNA pool was used for amplification of all tested transcripts. The relative quantification software (Bio-Rad) was used to determine the efficiency-corrected relative transcript concentration, normalized to a calibrator sample (Radutoiu et al., 2003). ATP synthase was used as a reference gene (AW719841). Melting curve analysis and sequencing of the amplified products was used to determine the identity of the amplified PCR products. Intron spanning primers were used for transcript amplification of the ATP synthase (5′-CAATGTCGCCAAGGCCCATGGTG-3′ and 5′- AACACCACTCTCGATCATTTCTCTG-3′), SIP1 (5′-CTTGCAGGTAAGTTCACAAC-3′ and 5′-GTACGAGTCCAATCAGATCCAG-3′), and NIN (5′-AATGCTCTTGATCAGGCTG-3′ and 5′-AGGAGCCCAAGTGAGTGCTA-3′) genes. The same experiment was performed three times, and the averages of measurements were presented.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number EU559710.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Full-length cDNA sequence of L. japonicus SIP1.

ACKNOWLEDGMENTS

We thank Drs. Yongjun Lin, Chunli Chen, and Ms. Huazhi Song for advice on transient expression and confocal laser-scanning microscope. We also thank Dr. Allan Caplan for critical comments on the manuscript.

Received March 17, 2008; accepted June 13, 2008; published July 16, 2008.

1 This work was supported by the National Natural Science Foundation of China (grant no. 30570056 to Z.Z.), by the National Basic Research Program of China (grant no. 01CB108901 to Z.Z.), and by the National Science Foundation (grant nos. NSF-MCB 0548525 and NSF-IOB 0543923 to Z.H.).

[C] Some figures in this article are displayed in color online but in black and white in the print edition.

[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.108.119164

LITERATURE CITED

Arrighi JF, Barre A, Ben Amor B, Bersoult A, Soriano LC, Mirabella R, de Carvalho-Niebel F, Journet EP, Gherardi M, Huguet T, et al (2006) The Medicago truncatula lysine motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes. Plant Physiol 142: 265-279

Borisov AY, Madsen LH, Tsyganov VE, Umehara Y, Voroshilova VA, Batagov AO, Sandal N, Mortensen A, Schauser L, Ellis N, et al (2003) The Sym35 gene required for root nodule development in pea is an ortholog of NIN from Lotus japonicus. Plant Physiol 131: 1009-1017 Denarie J, Debelle F, Prome JC (1996) Rhizobium lipo- chitooligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis. Annu Rev Biochem 65: 503- 558

Ding Z, Gillespie L, Pate G (2003) Human MI-ER1 alpha and beta function as transcriptional repressors by recruitment of histone deacetylase 1 to their conserved ELM2 domain. Mol Cell Biol 23: 250- 258

Ehrhardt DW, Atkinson EM, Long SR (1992) Depolarization of alfalfa root hair membrane potential by Rhizobium meliloti Nod factors. Science 256: 998-1000

Ehrhardt DW, Wais R, Long SR (1996) Calcium spiking in plant root hairs responding to Rhizobium nodulation signals. Cell 85: 673-681

Endre G, Kereszt A, Kevei Z,Mihacea S, Kalo P, Kiss GB (2002) A receptor kinase gene regulating symbiosis nodule development. Nature 417: 962-966

Fahraeus A (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol 16: 374-381

Felle HH, Kondorosi E, Kondorosi A, Schultze M (1995) Nod signalinduced plasma membrane potential changes in alfalfa root hairs are differentially sensitive to structural modifications of the lipochitooligosaccharide. Plant J 7: 939-947

Felle HH, Kondorosi E, Kondorosi A, Schultze M (1996) Rapid alkalinazation in alfalfa root hairs in response to rhizobia lipochitooligosaccharide signals. Plant J 10: 295-301

Gleason C, Chaudhuri S, Yang T, Munoz A, Poovaiah BW, Oldroyd GE (2006) Nodulation independent of rhizobia induced by a calciumactivated kinase lacking autoinhibition. Nature 441: 1149- 1152

Gregory SL, Kortschak RD, Kalionis B, Saint R (1996) Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins. Mol Cell Biol 16: 792-799

Heckmann AB, Lombardo F,Miwa H, Perry JA, Bunnewell S, Parniske M, Wang TL, Downie JA (2006) Lotus japonicus nodulation requires two GRAS domain regulators, one of which is functionally conserved in a non-legume. Plant Physiol 142: 1739-1750

Herrscher RF, Kaplan MH, Lelsz DL, Das C, Scheuermann R, Tucker PW (1995) The immunoglobulin heavy-chain matrix-associating regions are bound by Bright: a B cell-specific trans-activator that describes a new DNA-binding protein family. Genes Dev 9: 3067-3082

Iwahara J, Clubb RT (1999) Solution structure of the DNA binding domain from Dead ringer, a sequence-specific AT-rich interaction domain (ARID). EMBO J 18: 6084-6094

Iwahara J, Iwahara M, Daughdrill GW, Ford J, Clubb RT (2002) The structure of the Dead ringer-DNA complex reveals how AT-rich interaction domains (ARIDs) recognize DNA. EMBO J 21: 1197-1209

Kevei Z, Lougnon G, Mergaert P, Horvath GV, Kereszt A, Jayaraman D, Zaman N, Marcel F, Regulski Z, Kiss GB, et al (2007) 3-Hydroxy-3- methylglutaryl coenzyme A reductase 1 interacts with NORK and is crucial for nodulation in Medicago truncatula. Plant Cell 19: 3974- 3989

Kistner C, Winzer T, Pitzschke A, Mulder L, Sato S, Kaneko T, Tabata S, Sandal N, Stougaard J, Parniske M (2005) Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis. Plant Cell 17: 2217- 2229

Kortschak RD, Tucker PW, Saint R (2000) ARID proteins come in from the desert. Trends Biochem Sci 25: 294-299

Kurkdjian AC (1995) Role of the differentiation of root epidermal cells in Nod factor (from Rhizobium meliloti) induced root hair depolarization of Medicago sativa. Plant Physiol 107: 783-790

Lerouge P, Roche P, Faucher C, Maillet F, Truchet G, Prome JC, Denarie J (1990) Symbiotic host-specificity of Rhizoium meliloti is determined by sulphated and acetylated glucosamine oligosaccharide signal. Nature 344: 781-784

Limpens E, Bisseling T (2003) Signaling in symbiosis. Curr Opin Plant Biol 6: 343-350

Limpens E, Franken C, Smit P, Willemse J, Bisseling T, Geurts R (2003) LysM domain receptor kinases regulating rhizobial Nod factor- induced infection. Science 302: 630-633

Ma L, Hong Z, Zhang Z (2007) Perinuclear and nuclear envelop localizations of Arabidopsis Ran proteins. Plant Cell Rep 26: 1373- 1382

Madsen EB, Madsen LH, Radutoiu S, Olbryt M, Rakwalska M, Szczyglowski K, Sato S, Kaneko T, Tabata S, Sandal N, et al (2003) A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature 425: 637-640

Margaert P, Van Montagu M, Prome JC, Holsters M (1993) Three unusual modifications, a D-arabinosyl, an N-methyl, and a carbamoyl group, are present on the Nod factors of Azorhizobium caulinodans strain ORS571. Proc Natl Acad Sci USA 90: 1551-1555

Miller J (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 352-355

Nixon JC, Rajaiya J, Webb CF (2004) Characterization of dominant negative forms of the transcription factor, Bright. J Biol Chem 279: 52465-52472

Oldroyd GED, Dowine JA (2004) Calcium kinase and nodulation signaling in legumes. Nat RevMol Cell Biol 5: 566-576

Peck MC, Fisher RF, Long SR (2006) Diverse flavonoids stimulate NodD1 binding to nod gene promoters in Sinorhizobium meliloti. J Bacteriol 188: 5417-5427

Radutoiu S,Madsen LH, Madsen EB, Felle HH, Umehara Y, Gronlund M, Sato S, Nakamura Y, Tabata S, Sandal N, et al (2003) Plant recognition of symbiotic bacteria requires two LysM receptor-like kinase. Nature 425: 585-592

Radutoiu S, Madsen LH, Madsen EB, Jurkiewicz A, Fukai E, Quistgaard EM, Albrektsen AS, James EK, Thirup S, Stougaard J (2007) LysM domains mediate lipochitin-oligosaccharide recognition and Nfr genes extend the symbiotic host range. EMBO J 26: 3923-3935

Ramon-Maiques S, Kuo AJ, Carney D, Matthews AG, Oettinger MA, Gozani O, Yang W (2007) The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2. Proc Natl Acad Sci USA 104: 18993-18998

Relic B, Talmont F, Kopcinska J, Golinowski W, Prome JC, Broughton WJ (1993) Biological activity of Rhizobium sp. NGR234 Nod- factors on Macroptilium atropurpureum. Mol Plant Microbe Interact 6: 764-774

Sanjuan J, Carlson RW, Spaink HP, Bhat RU, Barbour MW, Glushka J, Stacey G (1992) A 2-O-methylfucose moiety is present in the lipooligosaccharide nodulation signal of Bradyrhizobium japonicu. Proc Natl Acad Sci USA 89: 8789-8793

Scharf KD, Siddique M, Vierling E (2001) The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing alpha-crystallin domains (Acd proteins). Cell Stress Chaperones 6: 225-237

Schauser L, Roussis A, Stiller J, Stougaard J (1999) A plant regulator controlling development of symbiotic root nodules. Nature 402: 191-195

Shiu SH, Karlowski WM, Pan R, Tzeng YH, Mayer KFX, Li WH (2004) Comparative analysis of the receptor-like kinase family in Arabidopsis and rice. Plant Cell 16: 1220-1234

Smit P, Raedts J, Portyanko V, Debelle? F, Gough C, Bisseling T, Geurts R (2005) NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription. Science 308: 1789-1791

Spaink HP, Sheeley DM, van Brussel AA, Glushka J, York WS, Tak T, Geiger O, Kennedy EP, Reinhold VN, Lugtenberg BJ (1991) A novel highly unsaturated fatty acid moiety of lipo-oligosaccharide signals determines host specificity of Rhizobium. Nature 354: 125-130

Steen A, Buist G, Horsburgh GJ, Venema G, Kuipers OP, Foster SJ, Kok J (2005) AcmA of Lactococcus lactis is an N- acetylglucosamininidase with an optimal number of LysMdomains for proper functioning. FEBS J 272: 2854-2868

Steen A, Buist G, Leenhouts KJ, El Khattabi M, Grijpstra F, Zomer AL, Venema G, Kuipers OP, Kok J (2003) Cell wall attachment of a widely distributed peptidoglycan domain is hindered by cell wall constituents. J Biol Chem 278: 23874-23881

Stracke S, Kistner C, Yoshida S, Mulder L, Sato S, Kaneko T, Sandal N, Stougaard J, Szczyglowski K, Parniske M (2002) A plant receptorlike kinase required for both fugal and bacterial symbiosis. Nature 417: 959-962

Stros M, Launholt D, Grasser KD (2007) The HMG-box: a versatile protein domain occurring in a wide variety of DNA-binding proteins. Cell Mol Life Sci 64: 2590-2606

Tan K, Shaw AL, Madsen B, Jensen K, Taylor-Papadimitriou J, Freemont PS (2003) Human PLU-1 has transcriptional repression properties and interacts with the developmental transcription factors BF-1 and PAX9. J Biol Chem 278: 20507-20513

Valentine SA, Chen G, Shandala T, Fernandez J, Mische S, Saint R, Courey AJ (1998) Dorsal-mediated repression requires the formation of a multiprotein repression complex at the ventral silencer. Mol Cell Biol 18: 6584-6594

Wang CH, Su LH, Sun CH (2007) A novel ARID/Bright-like protein involved in transcriptional activation of cyst wall protein 1 gene in Giardia lamblia. J Biol Chem 282: 8905-8914

Webb KJ, Skot L, Nicholson MN, Jorgensen B, Mizen S (2000) Mesorhizobium loti increases root-specific expression of a calcium- binding protein homologue identified by promoter tagging in Lotus japonicus. Mol Plant Microbe Interact 13: 606-616

Wilsker D, Patsialou A, Dallas PB, Moran E (2002) ARID proteins: a diverse family of DNA binding proteins implicated in the control of cell growth, differentiation, and development. Cell Growth Differ 146: 95-106

Wilsker D, Probst L, Wain HM, Maltais L, Tucker PW, Moran E (2005) Nomenclature of the ARID family of DNA-binding proteins. Genomics 86: 242-251

Yoshida S, Parniske M (2005) Regulation of plant symbiosis receptor kinase (SymRK) through serine and threonine phosphorylation. J Biol Chem 280: 9203-9209

Zhang Z, Hong Z, Verma DPS (2000) Phragmoplastin polymerizes into spiral coiled structures via intermolecular interaction of two selfassembly domains. J Biol Chem 275: 8779-8784

Zhang Z, Quick MK, Kanelakis KC, Gijzen M, Krishna P (2003) Characterization of a plant homolog of Hop, a cochaperone of Hsp90. Plant Physiol 103: 525-535 Hui Zhu, Tao Chen, Maosheng Zhu, Qing Fang, Heng Kang, Zonglie Hong, and Zhongming Zhang*

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China (H.Z., T.C., M.Z., Q.F., H.K., Z.Z.); and Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83844-3052 (Z.H.)

* Corresponding author; e-mail zmzhang@mail.hzau.edu.cn.

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: Zhongming Zhang (zmzhang@mail.hzau.edu.cn).

Copyright American Society of Plant Biologists Sep 2008

(c) 2008 Plant Physiology. Provided by ProQuest LLC. All rights Reserved.