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Antiquity and Function of CASTOR and POLLUX, the Twin Ion Channel-Encoding Genes Key to the Evolution of Root Symbioses in Plants^1,[W],[OA]

Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546

	ABSTRACT

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Root symbioses with arbuscular mycorrhizal fungi and rhizobial bacteria share a common signaling pathway in legumes. Among the common symbiosis genes are CASTOR and POLLUX, the twin homologous genes in Lotus japonicus that encode putative ion channel proteins. Here, we show that the orthologs of CASTOR and POLLUX are ubiquitously present and highly conserved in both legumes and nonlegumes. Using rice (Oryza sativa) as a study system, we employ reverse genetic tools (knockout mutants and RNA interference) to demonstrate that Os-CASTOR and Os-POLLUX are indispensable for mycorrhizal symbiosis in rice. Furthermore, a cross-species complementation test indicates that Os-POLLUX can restore nodulation, but not rhizobial infection, to a Medicago truncatula dmi1 mutant.

In addition to the AM symbiosis, certain members of the Eurosid I angiosperms can enter into a root symbiosis with soil bacteria (Soltis et al., 1995). The symbiosis culminates in the formation of the root nodule, an optimal microenvironment for bacteria to fix atmospheric nitrogen and for nutrient exchange between the symbiotic partners. The root nodule symbiosis takes place in two major forms, involving either the gram-negative bacteria of the family Rhizobiaceae, collectively called rhizobia, or the gram-positive Frankia bacteria that are filamentous actinomycetes (Pawlowski and Bisseling, 1996). The majority of leguminous plants as well as a nonleguminous plant, Parasponia, can interact with rhizobia, whereas a diverse group of nonleguminous plants, so called actinorhizal plants, are able to enter into an interaction with Frankia bacteria. The legume-rhizobia symbiosis is best characterized at the cellular and molecular levels. The interaction is set in motion by an intimate communication between the host and rhizobia (Long, 1996; Spaink, 2000). The legume roots secrete into the rhizosphere (iso)flavonoids that attract the rhizobia to the root and trigger the production of a bacterial signal known as Nod factor. Nod factors are chitin-like lipochitooligosaccharides that are essential for activating the host symbiotic responses, including an early ion influx, calcium spiking, root hair deformation and curling, transcriptional reprogramming of the host symbiotic genes, and cortical cell divisions, that ultimately result in the formation of the rhizobium-infected root nodules (for review, see Oldroyd and Downie, 2004, 2006, 2008; Stacey et al., 2006; Jones et al., 2007).

Although the AM and rhizobial symbioses are morphologically distinct, the two are mechanistically related in legumes. A number of legume genes that are required for nodulation also are essential for the AM interaction (Kistner et al., 2005). Moreover, a subset of the host genes that are induced during legume-rhizobia symbiosis also is up-regulated during AM symbioses (Albrecht et al., 1999; Kistner and Parniske, 2002). The overlap of the two symbiotic pathways has led to the hypothesis that the evolutionarily younger legume root nodule symbiosis may have evolved from the more ancient AM symbiosis (LaRue and Weeden, 1994; Gianinazzi-Pearson, 1996; Zhu et al., 2006).

In recent years, the development of genetic and genomic tools for the two model legumes Medicago truncatula and Lotus japonicus has greatly facilitated the cloning of genes required for root symbioses (Stacey et al., 2006). Analysis of these genes has begun to reveal the Nod factor and mycorrhizal signaling pathways. The Nod factors are likely perceived directly by the receptor-like kinases, such as Lj-NFR1/Mt-LYK3 and Lj-NFR5/Mt-NFP, that contain peptidoglycan-binding LysM domains in the extracellular region (Limpens et al., 2003a; Madsen et al., 2003; Radutoiu et al., 2003; Arrighi et al., 2006; Smit et al., 2007). Downstream of the Nod factor receptors are a set of proteins that play a dual role in AM and nodulation symbioses. These proteins include the Leu-rich-repeat receptor kinase Mt-DMI2/Ms-NORK/Lj-SYMRK/Ps-SYM19 (Endre et al., 2002; Stracke et al., 2002), the ion channel proteins Lj-CASTOR and Lj-POLLUX/Mt-DMI1/Ps-SYM8 (Ane et al., 2004; Imaizumi-Anraku et al., 2005; Edwards et al., 2007), the two nucleoporins Lj-NUP85 and Lj-NUP133 (Kanamori et al., 2006; Saito et al., 2007), the Ca²⁺/calmodulin-dependent protein kinase Mt-DMI3/Lj-CCaMK/Ps-SYM9 (Levy et al., 2004; Mitra et al., 2004; Tirichine et al., 2006), and the DMI3-interacting protein Mt-IPD3/Lj-CYCLOPS (Messinese et al., 2007; Chen et al., 2008; Yano et al., 2008). Except for Mt-DMI3 and Mt-IPD3, all of these common symbiosis components act upstream of the Nod factor-induced calcium spiking (Ehrhardt et al., 1996; Oldroyd and Downie, 2004, 2006). Mt-DMI3 and Mt-IPD3 function downstream of calcium spiking and are presumably responsible for decoding the calcium spiking signal (Oldroyd and Downie, 2006), which subsequently activates the nodulation-specific transcription factors, such as Mt-NSP1, Mt-NSP2, Mt-ERN1, and Lj-NIN, leading to transcriptional reprogramming of the host symbiotic genes (Schauser et al., 1999; Kalo et al., 2005; Smit et al., 2005; Middleton et al., 2007). In contrast to the Nod factor signaling, the mycorrhiza-specific signaling components beyond the common symbiosis pathway are largely unknown.

Intriguingly, all cloned legume symbiosis genes, including both the common symbiosis genes and genes only required for rhizobial symbiosis, have orthologs in nonlegumes (Zhu et al., 2006). This finding offers an opportunity to address the evolution of root symbioses in plants by characterizing ortholog functionality across the legume and nonlegume boundary. For example, the Lj-NFR1 ortholog in Arabidopsis (Arabidopsis thaliana; At1g21630) has been shown to be essential for chitin signaling, indicating an evolutionary relationship between chitin and Nod factor perception (Zhu et al., 2006; Miya et al., 2007; Wan et al., 2008). As part of our effort to address the function of nonlegume orthologs of legume genes required for root symbioses, we seek to determine whether the common symbiosis genes in legumes also are required for mycorrhizal symbioses in nonlegumes using rice (Oryza sativa) as a study model. We as well as others have demonstrated that Os-DMI3, the rice ortholog of Mt-DMI3/Lj-CCaMK/Ps-SYM9, is required for AM symbiosis in rice and able to complement a M. truncatula dmi3 mutant (Godfroy et al., 2006; Chen et al., 2007). Two recent studies revealed that the Mt-DMI2/Ms-NORK/Lj-SYMRK/Ps-SYM19 orthologs from the two actinorhizal plants, Casuarina glauca and Datisca glomerata, are essential for root symbioses with both AM fungi and Frankia bacteria (Gherbi et al., 2008; Markmann et al., 2008). Here, we extend these studies to include Os-CASTOR and Os-POLLUX, the rice orthologs of the twin common symbiosis genes Lj-CASTOR and Lj-POLLUX. We demonstrate that the rice orthologs of CASTOR and POLLUX, namely Os-CASTOR and Os-POLLUX, are indispensable for mycorrhizal symbiosis in rice and that Os-POLLUX can restore nodulation, but not rhizobial infection, to a M. truncatula dmi1 mutant.

	RESULTS AND DISCUSSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Antiquity and Evolution of the CASTOR and POLLUX Homologs in Plants

CASTOR and POLLUX are two homologous genes encoding putative ion channels that are components of the common symbiosis pathway in L. japonicus (Imaizumi-Anraku et al., 2005). The twin genes possibly evolved from an ancient gene duplication event that dated before the monocot-dicot divergence (Zhu et al., 2006; Fig. 1 ). Under this evolutionary scenario, the duplicated gene copies have degenerated to perform complementary rather than redundant functions. It was speculated that CASTOR and POLLUX, analogous to many other ion channels, may act as heteromultimeric complexes (Jiang et al., 2002; Imaizumi-Anraku et al., 2005). The fact that CASTOR and POLLUX homologs also are present in Physcomitrella patens (moss), a basal lineage of land plants that can establish root symbioses with AM fungi (Ane et al., 2004), suggests that the progenitor of CASTOR and POLLUX is ancestral to land plants.

View larger version (10K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Phylogenetic tree (unrooted) of CASTOR and POLLUX homologs in M. truncatula (Mt), L. japonicus (Lj), soybean (Gm), poplar (Pt), grapevine (Vv), Arabidopsis (At), rice (Os), sorghum (Sb), and maize (Zm). The tree was based on the C-terminal approximately 650 amino acids of the proteins. Sequence alignments were performed using ClustalX (Thompson et al., 1997) and manually curated. The tree was constructed by MEGA3.1 (Kumar et al., 2004), using the UPGMA method. Numbers below the branches represent the percentages of 1,000 bootstrap replications supporting the particular nodes.

The CASTOR and POLLUX homologs are highly conserved over the C termini of approximately 650 amino acids, with a sequence identity ranging from 69% to 98% depending on the phylogenetic distances between species (Table I ). As shown in Table I, the level of sequence identity between orthologs (80%–98%) is always higher than that between within-species paralogs (72%–76%). Moreover, multiple sequence alignments of the conserved regions revealed numerous amino acid residues that could discriminate between CASTOR and POLLUX orthologs (Supplemental Fig. S1), further supporting the bifurcation of the two orthologous groups in phylogenetic analysis (Fig. 1). In contrast to the C-terminal region, the N terminus of these proteins appears to evolve more rapidly. Within each of the CASTOR and POLLUX orthologous groups, the N-terminal sequences are conserved only between closely related species but highly diverged between distantly related species. Visual and in silico analysis using the SIMPLE algorithm (http://www.biochem.ucl.ac.uk/bsm/SIMPLE/) revealed that the N-terminal regions of CASTOR and POLLUX proteins are rich in simple sequence repeats encoded by simple sequence repeats at the DNA level (data not shown), which may have contributed to the fast-evolution feature of this region (Hancock and Simon, 2005).

We have selected rice as a model system to assess the function of nonlegume orthologs of the legume symbiosis genes because rice is a mycorrhizal plant with a completely sequenced genome and available resources for high-throughput reverse genetic analysis (Jeong et al., 2006; Chen et al., 2007; Miyao et al., 2007). Os-CASTOR and Os-POLLUX were identified as Os03g62650 and Os01g64980, respectively, in the rice genome (Nipponbare) based on The Institute for Genomic Research Rice Genome Annotation (http://www.tigr.org/tdb/e2k1/osa1/; Zhu et al., 2006). Alignment of full-length cDNAs with the genomic sequences revealed a gene structure of 12 exons for both genes (Fig. 2A ), which is conserved with their legume and nonlegume counterparts listed in Table I (Ane et al., 2004; Imaizumi-Anraku et al., 2005). Both Os-CASTOR and Os-POLLUX also produce transcript variants that result from alternative splicing (data not shown). Quantitative reverse transcription (qRT)-PCR analysis indicated that Os-CASTOR and Os-POLLUX are expressed in all tissues tested, including roots, leaves, stems, and panicles, and the expression levels in the root were not enhanced by mycorrhizal colonization (Fig. 2B). This observation was further supported by the analysis of the Rice Gene Index database (http://compbio.dfci.harvard.edu/tgi), from which the cognate expressed sequences of Os-CASTOR (TC285740) and Os-POLLUX (TC285508 and TC334749) were derived from cDNA libraries of various plant tissues. The expression pattern of Os-CASTOR and Os-POLLUX appears to be similar to that observed for Lj-CASTOR and Lj-POLLUX in L. japonicus (Imaizumi-Anraku et al., 2005) but in contrast to that of Mt-DMI1 (orthologous to Lj-POLLUX), which is predominantly expressed in M. truncatula roots (Ane et al., 2004).

View larger version (37K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Isolation and characterization of Tos17/T-DNA insertion mutants of Os-CASTOR and Os-POLLUX. A, Gene structures of Os-CASTOR and Os-POLLUX and the Tos17/T-DNA insertion sites. The exons and introns are indicated by boxes and lines, respectively. Insertion sites of Tos17/T-DNA are indicated. B, Os-CASTOR and Os-POLLUX expression levels in roots, stems, leaves, panicles, and mycorrhizal roots. Relative transcript abundance was determined by qRT-PCR and normalized against Os-ubiquitin1. Error bars represent SD values from three independent biological replications. C, Identification of homozygous (–/–) insertion mutants by PCR. Top, Identification of positive insertion plants (+/– or –/–) by PCR using a pair of Tos17/T-DNA- and gene-specific primers. Middle, PCR analysis to distinguish between homozygous (–/–) and heterozygous (+/–) mutant plants using a primer pair flanking the Tos17/T-DNA insertion site that allowed the amplification of only the wild-type (WT) allele under given PCR conditions. Bottom, RT-PCR analysis of Os-CASTOR and Os-POLLUX expression in the wild-type and mutant plants. The primer positions are indicated in A.

View larger version (40K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Sequences and domain structures of Os-CASTOR and Os-POLLUX. The characteristic motifs and domains are underlined, including the four transmembrane (TM) helices and the filter, the pore helix, the hinge, and the RCK (for regulation of conductance of K+) domain.

We searched the rice mutant databases for putative Tos17 and T-DNA insertion lines to be used for functional analysis of Os-CASTOR and Os-POLLUX in root symbioses (Jeong et al., 2006; Miyao et al., 2007). We identified one T-DNA insertion allele for Os-CASTOR in the genetic background of the japonica rice cv Hawyoung (line no. C04353), hereafter referred to as Os-castor-1 (Jeon et al., 2000; Jeong et al., 2002). In the Os-castor-1 mutant, the T-DNA was inserted into the second 72-bp exon (Fig. 2A). For Os-POLLUX, we obtained two mutant alleles, named Os-pollux-1 and Os-pollux-2 (Fig. 2A). The Os-pollux-1 mutant was a T-DNA insertion line in the genetic background of cv Dongjin (line no. B02432), in which the T-DNA was inserted into the first intron. Os-pollux-2 was a Tos17 insertion line in the genotype Nipponbare derived from tissue culture (line no. NC6423). In Os-pollux-2, the retrotransposon Tos17 was inserted into the third exon. For all three mutants, the expression of Os-CASTOR or Os-POLLUX was disrupted based on RT-PCR analyses (Fig. 2C, bottom).

From progeny of each primary mutant line, positive T-DNA/Tos17 insertion plants were identified by PCR analysis using a pair of T-DNA/Tos17- and gene-specific primers (Fig. 2C, top). A second round of PCR analysis followed to discriminate between homozygous mutant (–/–) and heterozygous (+/–) plants using a primer pair flanking the T-DNA/Tos17 insertion sites that enabled the amplification of only the wild-type alleles under given PCR conditions (Fig. 2C, middle). Since T-DNA and Tos17 mutant lines may comprise multiple insertion sites, the wild-type plants segregated from the progeny of the heterozygous mutant lines were used as additional controls for the experiments described below.

Os-castor and Os-pollux Mutant Plants Are Defective in Mycorrhizal Symbiosis

To test whether Os-CASTOR and Os-POLLUX are required for AM symbiosis in rice, we inoculated the mutant and wild-type rice roots with the fungus Glomus intraradices. At 35 d after inoculation, wild-type plants were densely colonized by G. intraradices, exhibiting the range of symbiotic structures typical of a functional symbiosis, including intercellular and intracellular hyphae, vesicles, and arbuscules. In each of the 60 wild-type plants from the genotypes Nipponbare, Hawyoung, and Dongjin, approximately 60% to 85% of the total root length was colonized. Similar levels of colonization also were observed for wild-type plants segregated from heterozygous mutant plants (Fig. 4 ). In contrast, intracellular fungal structures, including vesicles and arbuscules, were not observed on roots of 60 Os-castor-1, 72 Os-pollux-1, and 60 Os-pollux-2 homozygous mutant plants. For homozygous mutant plants, extraradical hyphae and appressoria were frequently observed on the root surface (Fig. 4), but the fungus was unable to penetrate the roots beyond the epidermis. These observations indicate that the knockout of Os-CASTOR and Os-POLLUX has completely abolished the ability of the AM fungus to enter the plant root. The defective phenotypes were similar to those reported for the castor (i.e. Lj-sym4, Lj-sym22, and Lj-sym71; Bonfante et al., 2000; Senoo et al., 2000) and pollux (i.e. Lj-sym23 and Lj-sym86; Kistner et al., 2005) mutants in L. japonicus, the dmi1 mutants in M. truncatula (Catoira et al., 2000), and the sym8 mutant in pea (Pisum sativum; Balaji et al., 1994). For the weak alleles of the L. japonicus castor and pollux mutants, the fungus was occasionally able to penetrate the cortical cells and form arbuscules (Senoo et al., 2000; Kistner et al., 2005), but this leaky phenotype was not observed for the knockout lines of rice.

View larger version (91K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Os-castor and Os-pollux are defective in AM symbiosis. A to C, Roots of wild-type plants segregated from heterozygous mutants formed arbuscules upon inoculation with G. intraradices. D to F, Roots of homozygous mutants failed to form AM symbiosis, despite the presence of fungal hyphae on the root surface. Photographs were taken from roots at 5 weeks after inoculation with G. intraradices. Mycorrhizal colonization was assessed by trypan blue staining according to the procedures described by Koske and Gemma (1989). Stained roots were examined using a light microscope (Olympus BX40F-3), and images were captured by a microscope digital camera system (Olympus DP71). ap, Appressorium; ar, arbuscule; eh, extraradical hypha; ih, intraradical hypha.

View larger version (29K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Expression of the rice AM-specific phosphate transporter Os-PT11 in the mutant and/or RNAi lines of Os-CASTOR and Os-POLLUX under inoculated and noninoculated conditions. A, Expression of Os-PT11 in the wild-type (WT) and mutant roots under inoculated and noninoculated conditions. WT* indicates wild-type plants segregated from a corresponding heterozygous mutant plant. B, Expression of Os-PT11 in the two RNAi lines, Os-CASTORi-1 and Os-CASTORi-2, during AM symbiosis. Numbers in parentheses indicate cycle numbers of the RT-PCR. The rice Actin gene was used as a control.

Os-POLLUX Can Restore Nodulation, But Not Rhizobial Infection, to a M. truncatula dmi1 Mutant

To determine whether the nonlegume CASTOR/POLLUX orthologs possess an equivalent function to their legume counterparts, we introduced two Os-POLLUX full-length cDNAs (AK067564 and AK073102), under the control of the 35S promoter, into the M. truncatula dmi1-1 mutant (allele C71; Catoira et al., 2000; Ane et al., 2004) using Agrobacterium rhizogenes-mediated hairy root transformation (Boisson-Dernier et al., 2001). AK067564 and AK073162, differing at their 3' ends due to alternative splicing, encode predicted proteins of 943 and 965 amino acids, respectively, of which the 943-amino acid version is canonical based on sequence alignments with other POLLUX orthologs. Nevertheless, the first 941 amino acids are shared between the two isoforms. It is noteworthy that both cDNA clones provided by the Rice Genome Resource Center in Japan contain a point mutation likely resulting from the RT process: there was an insertion of T at position 2,136 of AK067564 and an A-to-T transversion at position 593 of AK073012. Both errors were corrected for the transformation experiments described below. We did not observe any obvious differences resulting from using the two constructs, suggesting that the two protein isoforms are equally functional.

Upon inoculation with Sinorhizobium meliloti, the M. truncatula dmi1-1 mutant fails to exhibit root hair curling, infection thread formation, cortical cell division, and nodule development (Catoira et al., 2000). To determine whether Os-POLLUX was able to complement the nonnodulation phenotype of the dmi1-1 mutant, we inoculated the composite transgenic plants with S. meliloti strain 2011 carrying the lacZ reporter gene in plasmid pXLGD4 (Catoira et al., 2000). Two weeks after inoculation, nodule-like organs were observed on roots of 36 of 59 dmi1-1 plants transformed with Os-POLLUX. However, unlike wild-type plants, which form long, pink, cylindrical nodules, the transgenic mutant roots produce small, white, round nodules that are similar to the phenotypes of the nitrogen fixation mutants of M. truncatula (Starker et al., 2006). GUS staining and microscopy of sectioned nodules revealed that the small white nodules were devoid of bacteria and no infection threads were observed; thus, these nodules were nonfunctional (Fig. 6 ). The lack of nitrogen-fixing ability of these white nodules also was reflected by the poor growth and chlorotic leaves of the composite transgenic plants under nitrogen-starving conditions. Although Os-POLLUX is required for AM symbiosis in rice, the 35S:Os-POLLUX failed to complement the M. truncatula dmi1-1 mutant for AM symbiosis (data not shown).

View larger version (92K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Complementation of the M. truncatula dmi1-1 mutant (C71) with Os-POLLUX using A. rhizogenes-mediated hairy root transformation (Boisson-Dernier et al., 2001). A full-length cDNA clone of Os-POLLUX (AK067564) was cloned into a binary vector modified from pHellsgate8 driven by the 35S promoter (Helliwell et al., 2002). The binary vector was introduced into the A. rhizogenes strain, ARqua1, and transformed into the roots of the dmi1-1 mutant. A to D, Wild type. E to H, The dmi1-1 mutant transformed with 35S:Os-POLLUX. A and E, Nodule phenotypes of wild-type and transgenic dmi1-1 roots, respectively. B and F, GUS staining of nodules shown in A and E, respectively. C and G, Toluidine blue staining of sectioned nodules from wild-type and transgenic roots, respectively. D and H, Close-ups of the sectioned nodules.

	CONCLUSION

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Root symbioses with AM fungi and nitrogen-fixing bacteria share common signaling components, suggesting that the nitrogen-fixing root nodule symbioses have evolved from the ancient AM symbiosis (Kistner and Parniske, 2002). This hypothesis is further supported by the fact that all of the legume common symbiosis genes are present in nonlegumes that have the ability to establish the AM symbiosis (Zhu et al., 2006). However, the function of these nonlegume orthologs needs to be addressed, in order to gain insight into the evolution of the root symbioses in plants. Thus far, five of the seven known common symbiosis genes have been shown to be required for AM symbiosis in nonlegumes (Chen et al., 2007, 2008; Banba et al., 2008; Gherbi et al., 2008; Markmann et al., 2008). We speculate that all common symbiosis genes in legumes would be required for the AM symbiosis in nonlegumes. Interestingly, those genes required only for rhizobial symbiosis but not essential for the AM symbiosis in legumes are also present in nonlegumes. Elucidation of the function of nonlegume orthologs of nodulation-specific genes will provide further insights into the evolution of root symbioses in land plants.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Rice and Medicago truncatula Mutants

The rice (Oryza sativa) Tos17 insertion line (NC6423) in the Nipponbare background was provided by the Rice Genome Resource Center of the National Institute of Agrobiological Sciences in Japan. The rice T-DNA insertion mutants, C04353 and B02432, in the genetic backgrounds of Hawyoung and Dongjin, respectively, were provided by the Pohang University of Science and Technology in Korea. The Medicago truncatula dmi1-1 mutant (C71) was obtained from Dr. Douglas Cook's laboratory at the University of California, Davis.

Isolation of Homozygous Os-castor and Os-pollux Mutant Lines

Seeds of the Tos17/T-DNA insertion lines from the providers were from the progeny of primary transgenic or tissue culture-derived plants. To isolate homozygous mutants, we carried out two rounds of PCR analyses. The first round of PCR was to identify plants with Tos17/T-DNA insertion using the Tos17/T-DNA-specific and Os-CASTOR/Os-POLLUX-specific primer pairs. The second round of PCR was conducted to identify homozygous mutant plants using the primer pairs flanking the putative Tos17/T-DNA insertion sites. The positions of these primers are indicated in Figure 2A. Primer sequences are as follow: F1, 5'-CGATGGTCAGGGATGGTATC-3'; R1, 5'-CGTGTGGCTTTGCTCTATGA-3'; F2, 5'-CGATTTGATCTCTCCCCGTA-3'; R2, 5'-GCTGACAACATAAAGCGCAA-3'; T1, 5'-ACGCTGAACTTGTGGCCGTT-3'; T2, 5'-CCACAGTTTTCGCGATCCAGACTG-3'; T3, 5'-ATTGTTAGGTTGCAAGTTAGTTAAGA-3'.

Binary Vector Construction and Rice Transformation

For the generation of RNAi knockdown lines of Os-CASTOR, a 400-bp inverted-repeat sequence from the third exon of Os-CASTOR was cloned into the Gateway-enabled pSTARGATE vector (provided by CSIRO Plant Industry in Australia). The expression of the inverted-repeat sequence in pSTARGATE was driven by a ubiquitin promoter. The construct was introduced into Agrobacterium tumefaciens strain EHA105 and transformed to Nipponbare as described by Hiei et al. (1994). Primary transgenic plants (T0) were selfed to obtain the T1 generation for phenotypic analysis of AM symbiosis.

Hairy Root Transformation of M. truncatula

The dmi1-1 mutant of M. truncatula was transformed with Os-POLLUX using Agrobacterium rhizogenes-mediated hairy root transformation (Boisson-Dernier et al., 2001). Two full-length cDNA clones of Os-POLLUX (AK067564 and AK073162) were cloned into a binary vector modified from pHellsgate8 driven by the 35S promoter (Helliwell et al., 2002). The binary vector was introduced into the A. rhizogenes strain ARqua1 and transformed into the roots of the dmi1-1 mutant. Transformed roots were selected on Färhaeus medium (Färhaeus, 1957) containing 20 mg L^–1 kanamycin for 2 weeks at 20°C. The transgenic roots were further confirmed by PCR analysis.

Inoculation of Rice Roots with Glomus intraradices

The AM fungus Glomus intraradices was from Premier Tech Biotechnologies. The inoculation method was as described by Chen et al. (2007). Briefly, the rice plants were grown in 11-cm pots with sterilized Turface covered with 3 cm of sand in a growth chamber with a 13-h-light, 28°C/11-h-dark, 24°C regime. The plants were fertilized twice weekly with half-strength Hoagland solution (Arnon and Hoagland, 1940) supplemented with 100 µM KH₂PO₄. Roots of 2-week-old rice plants were inoculated by adding 1,000 spores to the sand at 1.5 cm depth. Roots were harvested at 5 weeks after inoculation. A random sample of the root tissues was used for phenotyping analysis, and the remaining tissues were used for RNA isolation.

Mycorrhizal colonization was phenotyped by means of trypan blue staining according to the protocol described by Koske and Gemma (1989). The cleaned roots were first fixed in 50% (v/v) ethanol. The fixed roots were then incubated at 90°C in 10% KOH for approximately 20 min. After rinsing with water, the roots were soaked in 1% HCl at room temperature overnight. The roots were then stained at 90°C for 30 min in an acidic glycerol solution containing 0.1% trypan blue. After destaining in acidic glycerol, the roots were examined using a light microscope (Olympus BX40F-3), and images were captured by a microscope digital camera system (Olympus DP71).

Inoculation of A. rhizogenes-Transformed M. truncatula Roots with Sinorhizobium meliloti

The nodulation assay was conducted as described by Limpens et al. (2003b). Three weeks after transformation, composite plants were transferred to sterile Turface saturated with Färhaeus medium [without Ca(NO₃)₂] for 3 d at 21°C and 16/8 h of light/dark. Each plant was then inoculated with 1 mL of culture (optical density at 600 nm = 0.1) of Sinorhizobium meliloti strain 2011 carrying the lacZ reporter gene in plasmid pXLGD4 (Catoira et al., 2000). The nodulation was scored 2 weeks after inoculation.

Nodule Sectioning, Staining, and Microscopy

Nodules were stained with X-gal to detect the presence of bacteria. For nodule sectioning, nodules were cut in half longitudinally, placed in FAA solution (100 mL: 45 mL of 95% ethanol, 40 mL of distilled water, 5 mL of glacial acetic acid, and 10 mL of 37% [w/w] formaldehyde) and vacuum infiltrated until they sank. The FAA fixation was followed by several steps of ethanol dehydration (50%, 60%, 70%, 80%, 95%, and two changes of 100% ethanol each for 30 min). The samples were then gradually infiltrated with Hemo-De (20%, 50%, and 75% Hemo-De solutions, each for 30 min, then two changes of 100% Hemo-De each for 1 h). Once hydrated with Hemo-De, the samples were infiltrated with Paraplast Plus by successively adding chips of Paraplast to Hemo-De at 42°C. After removing the Paraplast/Hemo-De solution, melted Paraplast was added and incubated at 60°C for at least 8 h. (This step was repeated for at least six changes of Paraplast.) The samples were then embedded and sectioned. The samples were sectioned with a Leica RM2135 microtome.

For light microscopy, 5-µm-thick sections were dried onto glass slides. The slides went through stepwise de-Paraplast and hydration and were stained with 1% (w/v) toluidine blue in 95% ethanol. Photographs were taken with an Olympus BX40F-3 light microscope, and images were captured by an Olympus DP71 microscope digital camera system.

Analysis of Gene Expression

Total RNA was isolated by the Qiagen Plant RNeasy kit. Two micrograms of RNA was used to perform RT reactions using M-MLV reverse transcriptase (Invitrogen) in a 20-µL reaction mixture. Two microliters of the RT reaction was used as a template in a 20-µL PCR solution. The PCR primers were as follows: Os-Actin, 5'-GCGATAATGGAACTGGTATG-3' and 5'-CTCCATTTCCTGGTCATAGTC-3'; Os-CASTOR, 5'-CGATGGTCAGGGATGGTATC-3' and 5'-CGTGTGGCTTTGCTCTATGA-3'; Os-POLLUX, 5'-CGATTTGATCTCTCCCCGTA-3' and 5'-GCTGACAACATAAAGCGCAA-3'; Os-PT11, 5'-ATGGCTCGACGGACAGTAAG-3' and 5'-GATCAGCTGGATCATGTACCT-3'. qRT-PCR was performed on the Applied Biosystems StepOne Real-Time PCR System using the SYBR Green I detection kit (Bio-Rad). The Os-ubiquitin gene was selected as a constitutive internal control. PCR primers used for the real-time PCR experiments were as follows: Os-ubiquitin, 5'-TGCACCCTAGGGCTGTCAAC-3' and 5'-TGACGCTCTAGTTCTTGATCTTCTTC-3'; Os-CASTOR, 5'-CAAGAGGGTGATGAGGTGCTAGTA-3' and 5'-GGTAACCTCTCATAACCTTGGGTAAT-3'; Os-POLLUX, 5'-CCTCGGATGGAGCGACAA-3' and 5'-ACGACACCACCACCAATACTCTT-3'.

Supplemental Data

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

Supplemental Figure S1. Sequence alignment of the CASTOR and POLLUX homologs.

Supplemental Sequence Data S1. The amino acid sequences of the CASTOR and POLLUX homologs.

	ACKNOWLEDGMENTS

 
We thank Dr. Douglas Cook for providing seeds of the M. truncatula mutants, the Rice Genome Resource Center for providing the rice Tos17 mutant lines and full-length cDNA clones, and the Pohang University of Science and Technology for providing the rice T-DNA insertion mutants. We also thank Dr. Sharyn Perry for help with nodule sectioning.

Received October 21, 2008; accepted October 28, 2008; published October 31, 2008.

	FOOTNOTES

 
¹ This work was supported by the Kentucky Science and Engineering Foundation (grant to H.Z.) and by the U.S. National Science Foundation (grant no. IOS 0640197 to H.Z.).

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: Hongyan Zhu (hzhu4{at}uky.edu).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.131540

^* Corresponding author; e-mail hzhu4{at}uky.edu.

	LITERATURE CITED

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