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First published online January 19, 2007; 10.1104/pp.106.092494 Plant Physiology 144:682-694 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
An IRE-Like AGC Kinase Gene, MtIRE, Has Unique Expression in the Invasion Zone of Developing Root Nodules in Medicago truncatula1,[OA]University of North Texas, Department of Biological Sciences, Denton, Texas 76203–5220
The AGC protein kinase family (cAMP-dependent protein kinases A, cGMP-dependent protein kinases G, and phospholipid-dependent protein kinases C) have important roles regulating growth and development in animals and fungi. They are activated via lipid second messengers by 3-phosphoinositide-dependent protein kinase coupling lipid signals to phosphorylation of the AGC kinases. These phosphorylate downstream signal transduction protein targets. AGC kinases are becoming better studied in plants, especially in Arabidopsis (Arabidopsis thaliana), where specific AGC kinases have been shown to have key roles in regulating growth signal pathways. We report here the isolation and characterization of the first AGC kinase gene identified in Medicago truncatula, MtIRE. It was cloned by homology with the Arabidopsis INCOMPLETE ROOT HAIR ELONGATION (IRE) gene. Semiquantitative reverse transcription-polymerase chain reaction analysis shows that, unlike its Arabidopsis counterpart, MtIRE is not expressed in uninoculated roots, but is expressed in root systems that have been inoculated with Sinorhizobium meliloti and are developing root nodules. MtIRE expression is also found in flowers. Expression analysis of a time course of nodule development and of nodulating root systems of many Medicago nodulation mutants shows MtIRE expression correlates with infected cell maturation during nodule development. During the course of these experiments, nine Medicago nodulation mutants, including sli and dnf1 to 7 mutants, were evaluated for the first time for their microscopic nodule phenotype using S. meliloti constitutively expressing lacZ. Spatial localization of a pMtIRE-gusA transgene in transformed roots of composite plants showed that MtIRE expression is confined to the proximal part of the invasion zone, zone II, found in indeterminate nodules. This suggests MtIRE is useful as an expression marker for this region of the invasion zone.
Nitrogen-fixing root nodules are the result of a complex and unique interaction between leguminous plants and soil rhizobia. During nodule development, rhizobia are brought into roots through infection threads that originate in deformed root hairs that curl to form a so-called "shepherd's crook." Infection thread initiation and growth require living rhizobia that are synthesizing specific Nod factors (Ardourel et al., 1994  ; Limpens et al., 2003). Infection threads bring rhizobia past several root cell layers and deposit them into newly divided nodule cells within a membrane-bounded compartment called the symbiosome in a process resembling endocytosis. Within the infected cells, an environment is established where the rhizobia and plant express new proteins that enable biochemical support of nitrogen fixation and assimilation. In indeterminate nodulators such as Medicago truncatula, new infections occur continuously over the lifetime of the nodule, with new infections starting below the meristematic distal end of the nodule at the beginning of the infection zone, zone II. In the proximal end of zone II, both rhizobia and plant cells expand and mature. Exit from zone II is marked by starch accumulation, which the rhizobia apparently use as a carbon source as they continue their maturation to nitrogen fixation capability in zone III (Vasse et al., 1990). Reviews on legume root nodule development are available (Brewin, 1991; Kijne, 1992; Gage and Margolin, 2000; Brewin, 2004; Gage, 2004).
Nodule-specific (nodulin) and nodule-enhanced genes are expressed exclusively and primarily in nodules, respectively. Most nodulin genes have homologs in nonlegumes, suggesting that nodule-specific genes have been recruited from other developmental pathways. It has been noted that a number of nodulins are also expressed in nonsymbiotic tissue, including in floral tissues (Szczyglowski and Amyot, 2003
The AGC protein kinases contain the protein kinase A, protein kinase G, and protein kinase C regulatory kinases. In animals and fungi, members of this kinase family act in protein phosphorylation cascades. In animals, a key AGC kinase regulator is the 3-phosphoinositide-dependent protein kinase (PDK1), a central growth regulator that integrates signaling events from receptors that stimulate the synthesis of phosphatidylinositol 3,4,5-trisphosphate (Bogre et al., 2003
The Medicago genome project has identified many potential genes with roles in nodule development by virtue of their being found as expressed tag sequences (ESTs) only in cDNA libraries prepared from tissue that includes nodules (Fedorova et al., 2002
Isolation and Sequence of the Complete MtIRE Gene
In 2002, Fedorova et al. reported 340 tentative consensus (TC) sequences comprising ESTs found only in cDNA libraries that were made from M. truncatula nodule-containing tissue (Fedorova et al., 2002
The Medicago genome project has mapped BACs mth2-13b8 and mth1-8d23 containing MtIRE to chromosome 5 (www.medicago.org/genome). Comparison of the MtIRE genomic and cDNAs revealed that the MtIRE gene spans a genomic region of 9.1 kb and consists of 17 exons and 16 introns (Fig. 1A ). This organization is almost identical to the AtIRE gene, which is shown for comparison in Figure 1B. Both genes have 17 exons of similar sizes, but in the Medicago MtIRE gene the introns are significantly larger than in the AtIRE gene. The Arabidopsis genome has four other IRE-like genes. Their genome organization is similar to AtIRE and to MtIRE although slight differences are noted, especially extra exons in AtIRE_2 and the lack of the first three exons in AtIRE_3 (Fig. 1C).
The third start codon (ATG) from the 5' end of the transcript is the presumed start site for the longest open reading frame of the MtIRE gene. MtIRE encodes a deduced protein of 1,168 amino acid residues. ExPASy (www.expasy.org/tools/scanprosite; Gattiker et al., 2002 ) and BLAST (www.ncbi.nlm.nih.gov/BLAST/; Altschul et al., 1990) tools were used to analyze the deduced MtIRE protein (Fig. 1D). It contains a Glu-rich region at its N-terminal end, basic-type (Kalderon et al., 1984) and bipartite-type (Robbins et al., 1991) nuclear localization signals, a zinc finger-like sequence (C-X2-C-X11-H-X3-C; Fig. 1D; Bohm et al., 1997), and numerous potential phosphorylation sites, including those for casein kinase II, protein kinase C, cAMP-, cGMP-, and phosphoinositide-dependent protein kinases. MtIRE contains a putative Ser/Thr protein kinase domain toward its C-terminal end (Fig. 1D). Within the Ser/Thr protein kinase domain is an activation loop motif that is a putative target of PDK1. C terminal to the kinase domain is the PDK1-interacting fragment (PIF) found in some PDK1 substrates (Fig. 1D). Both the activation loop motif and the PIF are signature sequences of the AGC family (Bogre et al., 2003; Mora et al., 2004).
BLAST (Altschul et al., 1990
MtIRE, IRE homologs, and the Arabidopsis AGC kinases (Bogre et al., 2003 ) were subjected to phylogenetic analysis using ClustalW (Thompson et al., 1994) and the neighbor-joining method. The Medicago IRE genes were found to group as a single clade with the AtIRE, rice OsIRE, and tomato LeIRE genes (Fig. 2
). This suggests that MtIRE, AtIRE, and other IRE-like genes diverged from the other AGC kinases before the monocots and dicots diverged, estimated to be about 170 million years ago (Sanderson et al., 2004). MtIRE was found to form a subclade with AtIRE within the IRE clade (Fig. 2), suggesting that these two genes are orthologous.
The N termini of MtIRE, AtIRE, and OsIRE are highly diverged (not shown), while the putative nuclear localization sequences, zinc finger, PIF motif, and Ser/Thr protein kinase domain are conserved (Fig. 3 ). Within the Ser/Thr protein kinase domain, the activation loop signature motif is conserved among the IRE genes, including MtIRE. However, the putative PDK1 kinase target Ser residue of the activation loop was found to be missing in both AtIRE and OsIRE; it is present in MtIRE (Fig. 3D) and also present in the other IRE genes for which full sequence is available (not shown).
MtIRE Is a Single Copy Gene in Medicago To experimentally determine the MtIRE copy number in M. truncatula, Southern-blot analysis was carried out. M. truncatula genomic DNA was restricted with several enzymes, blotted, and probed with a cDNA probes derived from exon 4 of MtIRE (Fig. 1). The results obtained at low stringency (not shown) are similar to those obtained at high stringency (Fig. 4 ), showing one or two bands per lane. These results are consistent with MtIRE being a single copy gene in M. truncatula, and, with the exception of the pattern obtained with HincII enzyme, consistent with predicted in silico restriction pattern. In some cases, HincII cleavage is affected by DNA methylation, which may account for the inconsistency with the in silico prediction. Probing the blot with a different cDNA derived from the putative Ser/Thr kinase domain (Fig. 1) yielded similar results of one or two bands per lane, also consistent with MtIRE being single copy (not shown).
MtIRE Expression Is in Nodules and Flowers To examine MtIRE expression, semiquantitative reverse transcription (RT)-PCR was carried out on total RNA extracted from different plant tissues of M. truncatula A17 wild-type plants. RT-PCR primers were chosen to flank introns (Fig. 1). The transcript of the MtIRE gene was detected only in nodules and flowers and not in other organs of the plant, including uninoculated roots (Fig. 5A ).
To determine when during nodule development MtIRE is first expressed, a time course of MtIRE expression in roots with developing nodules was carried out. The results show that MtIRE is first expressed at 4 d postinoculation (dpi) of nitrogen-starved roots with Sinorhizobium meliloti (Fig. 5B). This is well before the onset of nitrogen fixation in growth conditions used for these experiments, at about 8 dpi.
To further pin down the stage of nodule development with which MtIRE gene expression is associated, M. truncatula nodulation mutants were examined for MtIRE expression in nodulated root systems or root areas susceptible to nodulation 10 dpi after inoculation with S. meliloti, also by semiquantitative RT-PCR. As shown in Figure 6A
, MtIRE was found to be expressed in both supernodulators tested, sunn (Penmetsa et al., 2003
To correlate MtIRE expression in the mutant nodules with phenotypes of the mutants, microscopic analyses of mutant nodules were carried out by examining X-Gal-stained 50-µm sections of nodules or whole roots of mutant plants inoculated with S. meliloti/hemA::lacZ (Boivin et al., 1990 ), also at 10 dpi (Fig. 7
). While similar analyses have been reported for some of the mutants used in this study, it was important to verify the phenotypes under the growth conditions employed in these experiments.
As shown in Figure 7A, for the skl mutant, more nodules than wild type, all staining so darkly blue they almost look black, were detected, as reported previously (Penmetsa and Cook, 1997 ). For dmi2, no invasion was seen; as reported previously, root hairs developed bulbous tips (Catoira et al., 2000; Fig. 7B). For the four mutants with defects in rhizobial invasion, lin, sli, nip, and Mtsym1, a gradient of rhizobial infection was observed, with lin infections confined to the root hair cell containing the initial infection thread, similar to published observations (Kuppusamy et al., 2004; Fig. 7C). In the sli mutant, infections progressed further, with infection threads penetrating the outer cortical cells and occasionally reaching inner cortical cells (Fig. 7D). The rare invaded nodules seen previously (Haynes et al., 2004) at much later times postinoculation were not observed in our growth conditions at the 10-dpi time point. In the nip mutant, infection threads filled nodule primordia that showed signs of defense responses by accumulating brown pigments, as described previously (Veereshlingam et al., 2004; Fig. 7E). For Mtsym1 (Benaben et al., 1995; Fig. 7F), nodule primordia filled with infection threads were readily observed, as were nodules with rhizobia released into plant cells. Brown pigments were observed, although the accumulation of these pigments was less than in the nip mutant and were distributed differently from those in nip, mostly around infection threads in Mtsym1. These results are similar to those previously described by some researchers (Benaben et al., 1995). The small, uninvaded Mtsym1 bumps seen by others (Benaben et al., 1995; Mitra and Long, 2004) were only rarely observed in our conditions. The dnf mutants' nodules host cells all showed evidence of released rhizobia around a central vacuole (Fig. 7, G–R). Nodules from the dnf1 and dnf5 mutants appeared to have the most serious defects. dnf1-1 and dnf1-2 nodules were characterized by large accumulations of brown-orange pigments in the most proximal zone developed in these nodules. To a lesser extent, these pigments also accumulated in the nodule parenchyma (Fig. 7, G and H). In the dnf5 mutant, the rhizobia occupying infected cells appeared to be sparser than in wild-type nodules, and some accumulation of pigments was observed (Fig. 7N). The dnf2 mutant nodules appeared to have lower rhizobial occupancy but were clearly invaded by rhizobia. Only a small pale-yellow band that may be polyphenolic accumulation near the invasion zone was noted (Fig. 7I). For the dnf3, dnf4, dnf6, and dnf7 mutants, at least two different phenotype nodules were observed in our growth conditions. One type appeared defective with lower rhizobial occupancy in the infected cells (Fig. 7, J, L, O, and Q), while the second type was hard to distinguish from wild type (Fig. 7, K, M, P, and R). For the case of dnf7 mutants, nodulated root systems displayed only a few of the wild-type phenotype nodules (as in Fig. 7R). The defective dnf7 nodules look quite abnormal, with rhizobia accumulating in a band toward the apex of the nodule (Fig. 7Q).
To look more closely at mutant nodules not expressing MtIRE versus those with low levels of MtIRE expression, higher magnifications were used to compare the phenotypes of the nip and Mtsym1 nodules to those of dnf1 and dnf5 (Fig. 8
). Both nip and Mtsym1 nodules contain rhizobia mostly confined within infection threads (Fig. 8, A and B). At the subcellular level, both mutants have been shown by electron microscopy to have rhizobial release into host cells; in nip, this is a rare event, but it is frequent in Mtsym1 (Benaben et al., 1995
Spatial Localization of Nodule MtIRE Expression
To examine the spatial localization of MtIRE expression, an MtIRE promoter-GUS reporter construct (pMtIRE-gusA) was made and expressed in transgenic roots in composite M. truncatula wild-type plants. Plants were inoculated with rhizobia carrying a constitutive hemA gene, and nodulated roots were harvested at 15 dpi. Nodules formed asynchronously and more slowly in the composite plants than in untransformed roots, and nodules were found at various stages of development in the composite plants at 15 dpi in our growth conditions. Fixed tissue was stained with 5-bromo-4-chloro-3-indolyl-
Expression of the pMtIRE-gusA construct in composite dnf1-2 and dnf7 plants was evaluated to test the validity of using MtIRE as a marker for invaded nodule cell tissue. The results showed pMtIRE-gusA expression in both mutants as expected. dnf1-2 has smaller nodules than does dnf7 (and wild type) and pMtIRE-gusA staining was confined to the proximal end of the largest dnf1-2 nodules observed (Fig. 9D). For the largest observed dnf7 nodules, staining was observed in approximately the nodule middle (Fig. 9E), suggesting that dnf7 nodules are capable of further development than are those of dnf1-2. A pictorial interpretation of the visualized staining pattern for wild-type nodules is presented in Figure 9F. pMtIRE-gusA is confined to the proximal side of zone II, and thus may be a marker for this developmental zone.
In 2002, Fedorova et al. reported nodule-specific TC sequences found in Medicago. Among these was a TC with high homology for the AtIRE gene, with a predicted signal transduction function (Fedorova et al., 2002 ). Because the AtIRE gene has a known role in root hair elongation and is thought to function in regulating the duration of tip growth (Oyama et al., 2002), we thought that the MtIRE gene might have a similar role in Medicago or one regulating infection thread growth, which can be viewed as inward apical growth, similar to root hair tip or pollen tube growth.
MtIRE is a member of the AGC family of Ser/Thr protein kinases involved in signal transduction. In many animal AGC kinases, activation depends on sequential phosphorylation at two sites, one within the PIF motif, found C terminal to the kinase domain, and one within the activation loop of the kinase domain (Mora et al., 2004
The large plant AGC kinase family is subdivided into distinct phylogenetic classes (Bogre et al., 2003 Expression studies in developing nodules showed MtIRE expression is first detectable at 4 dpi in our growth conditions (Fig. 4). This time coincides with the time when symbiosomes are beginning to develop and is long before the onset of nitrogen fixation, at about 8 dpi in our conditions.
To correlate MtIRE expression with the phenotypes formed by nodulation mutants, it was necessary to define the mutated phenotypes in our growth conditions. Our findings show that at 10 dpi, sli nodules are blocked during invasion of the nodule primordia by rhizobia, different from the rare invaded nodules that are observed at a later time point (Haynes et al., 2004
Comparing MtIRE expression in mutant nodulating root systems (Fig. 6) with phenotypes of the mutant nodules (Figs. 7 and 8) shows that MtIRE expression is associated only with nodules that are able to achieve successful invasion, release of rhizobia into infected cells, and some development of the resulting symbiosomes, i.e. the dnf mutants. MtIRE expression was not observed in the sli or nip mutants that have little to no rhizobial release from infection threads (Haynes et al., 2004
In attempts to discern a function for MtIRE, we and others (S. Gantt, personal communication) have attempted MtIRE RNAi-induced gene silencing. The results have been inconsistent, with phenotypes that varied from root hair defects to defective nodulation to no detectable effect (data not shown). We have tried three different silencing constructs, with two chosen from MtIRE regions that are apparently unique to MtIRE and one from the putative Ser/Thr kinase domain. The lack of consistent results from any RNAi construct tested suggests possible functional redundancy in one or more of the other Medicago IRE-like genes. We note that another group has also reported similar issues with the RNAi technique (Liu et al., 2006
Localization experiments using pMtIRE-gusA in transformed hairy root composite plants showed that MtIRE expression localizes to a band in the proximal part of zone II, the infection zone. To date, there are very few details about the biology or biochemistry of the symbiotic interaction in this nodule region. In this area of zone II, rhizobia are already released from infection threads, symbiosomes are starting to develop and the host cells are expanding, but the rhizobia are not capable of nitrogen fixation (Vasse et al., 1990 MtIRE was found to be a single copy gene in M. truncatula and a member of the IRE clade of the AGC kinase family. The phylogenetic analysis suggests that MtIRE may be the ortholog of the AtIRE gene. We performed the phylogenetic analysis with the available, but partial, cDNA sequence for the three other Medicago IRE genes. When the full sequences of these cDNAs are available, it may turn out that one of them is a closer homolog to AtIRE than is MtIRE.
Our results demonstrated MtIRE expression in nodules and flowers (Fig. 3), similar to a number of other nodulins (Allison et al., 1993
Plants, Growth Conditions, and Rhizobial Strain
Medicago truncatula A17 (wild type) and nodulation mutants were grown in aeroponic chambers, starved for nitrogen for 5 d, and inoculated with Sinorhizobium meliloti harboring pXLDG4 (Boivin et al., 1990
RT-PCR was used to clone MtIRE from total RNA extracted from M. truncatula root nodules using standard protocols (Ausubel et al., 1988
ExPASy (www.expasy.org/tools/scanprosite; Gattiker et al., 2002
The MtIRE primers were designed to span introns: MtIRE F, 5'-CATCCATAAAGACCTAGGGGAAAAAGTTC; and MtIRE R, 5'-CTCCATGATTTCCTGCCCAAAGGC. The 18S rRNA F is 5'-CCAGGTCCAGACATAGTAAG and the 18S rRNA R is 5'-GTACAAAGGGCAGGGACGTA. Two micrograms of total RNA, extracted as described previously (Veereshlingam et al., 2004
Nodules elicited with S. meliloti/pXLDG4 containing the constitutive hemA::lacZ gene were stained with X-Gal, as described previously (Veereshlingam et al., 2004
pRD022 was created by subcloning the SphI fragment from pCAMBIA2301 (Hajdukiewicz et al., 1994
GUS activity in whole roots was detected after fixation in 0.3% paraformaldehyde and infiltration with X-gluc, as described by Jefferson (Jefferson et al., 1987 All experiments were done in at least duplicate. Sequence data for the genes discussed in this article can be found in the GenBank databases under the following accession numbers: MtIRE, AAX11214; AtIRE, NP_201037; AtIRE H1, NP_188412; AtIRE2, BAB02708; AtIRE3, NP_001031155; AtIRE4, NP_175130; OsIRE, ABA99908; OsIRE1, XP_469518; OsIRE2, ABF98517; OsIRE3, ABF98518; OsIRE4, AK122108; LeIRE, BT013855; AtPDK1_1, NP_974730; AtPDK1_2, ABF57283; AtS6K_2, NP_850543; AtS6K_1, NP_187485; AtNDR1, NP_849380; AtNDR2, NP_171888; AtNDR3, NP_188973; AtNDR4, NP_565453; AtNDR5, NP_179637; AtNDR6, NP_195034; AtNDR7, NP_174352; AtNDR8, NP_568221; AtAGC1_1, NP_200402; AtAGC1_2, NP_194391; AtAGC1_3, NP_850426; AtAGC1_4, NP_198819; AtAGC1_5, AAV85687; AtAGC1_6, NP_173094; AtAGC1_7, NP_178045; AtAGC1_8, NP_195984; AtAGC1_9, NP_181176; AtAGC1_10, NP_180238; AtAGC1_11, NP_188054; AtAGC1_12, NP_190047; AtPK3, NP_175774; AtPK5, NP_199586; AtPK7, AAQ65194; AtKIPK, NP_850687; AtPID, NP_181012; AtAGC2_1 (OXI1), NP_189162; AtAGC2_2, NP_193036; AtAGC2_3, NP_564584; AtAGC2_4, NP_188719; AtPHOT1, NP_190164; and AtPHOT2, NP_568874.
We thank several Medicago researchers for nodulation mutant seeds: Drs. Douglas Cook, Thierry Huguet, Sharon Long, Giles Oldroyd, Varma Penmetsa, Colby Starker, and Kate VandenBosch. We thank Heath Wessler for help with DNA sequencing, and Etienne Journet and David Barker for providing the pENOD11-gusA construct used as a control in root transformation and X-gluc staining experiments (not shown). We thank Ed Braun for helpful discussions about the phylogenetic analysis. Received November 1, 2006; accepted January 1, 2007; published January 19, 2007.
1 This work was supported by the National Science Foundation (IOB no. 0520728) and by the University of North Texas (faculty research funds to R.D.).  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: Rebecca Dickstein (beccad{at}unt.edu).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.092494 * Corresponding author; e-mail beccad{at}unt.edu; fax 940–565–3821.
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ABSTRACT
RESULTS
-glucuronic acid (X-gluc) to visualize pMtIRE-gusA. The largest wild-type nodules were costained with Red-Gal to visualize the rhizobia. The results (
