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First published online October 29, 2004; 10.1104/pp.104.045575 Plant Physiology 136:3682-3691 (2004) © 2004 American Society of Plant Biologists LIN, a Medicago truncatula Gene Required for Nodule Differentiation and Persistence of Rhizobial Infections1Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108 (K.T.K., G.E., K.A.VdB.); Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, H–6701 Szeged, Hungary (G.E.); Department of Biology, Texas A&M University, College Station, Texas 77843 (R.P.); Department of Plant Pathology, University of California, Davis, California 95616–8680 (V.P., D.R.C.); and Department of Biological Sciences, University of North Texas, Denton, Texas 76203–5220 (H.V., R.D.)
Ethyl methanesulfonate mutagenesis of the model legume Medicago truncatula has previously identified several genes required for early steps in nodulation. Here, we describe a new mutant that is defective in intermediate steps of nodule differentiation. The lin (lumpy infections) mutant is characterized by a 4-fold reduction in the number of infections, all of which arrest in the root epidermis, and by nodule primordia that initiate normally but fail to mature. Genetic analyses indicate that the symbiotic phenotype is conferred by a single gene that maps to the lower arm of linkage group 1. Transcriptional markers for early Nod factor responses (RIP1 and ENOD40) are induced in lin, as is another early nodulin, ENOD20, a gene expressed during the differentiation of nodule primordia. By contrast, other markers correlated with primordium differentiation (CCS52A), infection progression (MtN6), or nodule morphogenesis (ENOD2 and ENOD8) show reduced or no induction in homozygous lin individuals. Taken together, these results suggest that LIN functions in maintenance of rhizobial infections and differentiation of nodules from nodule primordia.
In response to specific soil bacteria called rhizobia, legume roots initiate a unique developmental program that culminates in the formation of nitrogen-fixing root nodules. The highly specialized microenvironment of the nodule provides conditions necessary for the conversion of atmospheric nitrogen to ammonium by the rhizobial nitrogenase enzyme. Nod factors, lipooligosaccharide signal molecules produced by the bacterial partner, are required for the parallel processes of bacterial infection and nodule morphogenesis (Geurts and Bisseling, 2002  ). In recent years, major advances have been made in defining the host's responses to Nod factors, culminating in the cloning of several plant genes required for Nod factor signal transduction (Schauser et al., 1999; Endre et al., 2002; Stracke et al., 2002; Limpens and Bisseling, 2003; Madsen et al., 2003; Radutoiu et al., 2003; Ané et al., 2004; Lévy et al., 2004). The characterization of these genes and their mutant phenotypes indicates that receptor-like kinases play multiple roles in the perception of Nod factor and that transduction of the signal proceeds via calcium-mediated responses and transcriptional regulation (Cullimore and Dénarié, 2003; Parniske and Downie, 2003; Riely et al., 2004).
Current understanding of the plant's contribution to symbiotic development subsequent to Nod factor perception, including rhizobial infection, primordium initiation, and nodule differentiation, comes largely from microscopic examination and characterization of plant genes that are differentially expressed during these processes. Early in the interaction, the bacteria gain access to the root through selective penetration of root hair cells. Invagination of the plant plasma membrane and deposition of cell wall material enable bacterial invasion via infection threads. Some, but not all, infection threads traverse the epidermal cells to invade root cortical cells that have been mitotically activated in response to Nod factor (Stougaard, 2000
Coincident with infection in the epidermis and outer cortical cells, cell divisions initiate in the root cortex, leading to the formation of a nodule primordium and ultimately to a differentiated nodule organ. In indeterminate nodules, such as those formed on alfalfa (Medicago sativa) and the model legume Medicago truncatula, some derivatives of the persistent meristem exit mitosis and enter a modified cell cycle characterized by endoreduplication, leading to an increase in ploidy and cell size (Cebolla et al., 1999 With the goal of identifying host genes that govern infection persistence, we screened ethyl methanesulfonate mutants of M. truncatula for mutants with an infection arrest phenotype. This study focuses on one such mutant, called lin (lumpy infections), that initiates but cannot sustain rhizobial infections. Although rhizobia elicit several Nod factor responses and nodule primordium formation on lin, the mutant is deficient in sustained meristematic activity. By investigating expression of genes that delineate stages of infection progression and primordium development, we demonstrate that lin is arrested prior to the onset of differentiation of nodule tissues. Genetic mapping has placed lin on linkage group 1 (LG1) of M. truncatula and fine mapping of the lin locus is under way. Thus, LIN defines a plant gene that is required for infection thread formation in root cortical cells and for formation of functional root nodules.
In an intensive screen for symbiotic mutants with altered nodulation phenotypes (Penmetsa and Cook, 2000 ), we identified a mutant line, C88, which developed small white bumps in response to rhizobia (Fig. 1, A–C). The phenotype of C88 was heritable in successive generations and was selected as a candidate for further analysis.
Observations made on stained whole mounts of fixed roots or root sections of mutant and wild-type plants showed aberrant development of infection threads in C88. Infection threads were first visible in the curled root hairs of both wild-type and mutant plants by 1 d postinoculation (dpi). In wild-type plants, the infection threads invaded the cortical layer by 3 dpi (Fig. 1, D and F). However, in C88, all infections arrested within root hairs, typically after very limited progression into the infected cell. Similar to wild-type plants, C88 plants displayed foci of cell divisions in the root cortex that formed the nascent nodule primordia. These developing primordia were not invaded by the infection threads and thus showed an arrest in differentiation (Fig. 1G) in comparison to wild-type nodules (Fig. 1F). As demonstrated in Figure 2, cellular differentiation was substantially reduced within C88 nodule primordia, although prevascular strands were sometimes noted to connect to the vascular cylinder of the root.
Infections in C88 occurred at less than one-fourth the frequency of infections in wild-type plants (Table I), and most infections occurred in coincidence with subtending nodule primordia. Interestingly, infection and formation of nodule primordia in C88 recurred continually during prolonged growth in the presence of rhizobium (Fig. 1, A–C). This phenotype is in contrast to wild-type plants, where initial infection and nodulation are transient, resulting in a narrow zone of primary nodule formation.
The Mutant Phenotype in C88 Is Conditioned by a Monogenic, Recessive Mutation That Maps to LG1 of the M. truncatula Map
For purposes of genetic characterization, C88 plants were crossed with the wild-type genotype A17. As shown in Table II, the frequency of nod+ and nod– phenotypes in the F2 generation was consistent with segregation as a single recessive allele. To determine whether C88 was allelic to other nodulation mutants, complementation analysis was performed with mutant lines C19 and C96, which showed an infection arrest phenotype, the production of small white nodules, or both. The F1 progeny from these crosses revealed that none of the lines tested was allelic to C88. C88, therefore, identifies a new complementation group. The gene conditioning the mutant phenotype in C88 was designated lin-1, for lumpy infections allele 1, in accordance with nomenclature rules for M. truncatula (VandenBosch and Frugoli, 2001
Mapping of lin was initiated to allow eventual map-based cloning of this gene. A mapping population was constructed by crossing lin in the background of the male sterile mutant tap with ecotype A20 (Penmetsa and Cook, 2000 ). The F1 progeny from the cross between lin and A20 showed a wild-type phenotype for nodulation, consistent with the recessive nature of the mutation. One hundred twenty-five F2 individuals were phenotyped for nodulation, with a segregation pattern consistent with the recessive nature of lin-1 (Table II).
Several molecular markers distributed on the eight linkage groups of the M. truncatula genetic map (Choi et al., 2004
The nodule-expressed gene MtN6 (Mathis et al., 1999 ) was also mapped to the lower arm of LG1 of M. truncatula (T. Huguet and P. Gamas, personal communication) based on a mapping population derived from the mapping parents Jemalong and DZA315 (Thoquet et al., 2002). Both the map location and the fact that MtN6 expression occurs spatially in advance of growing infection threads (Mathis et al., 1999) suggested that MtN6 and LIN could be the same gene. To test this hypothesis, we mapped MtN6 in the lin mapping population to examine the extent of cosegregation between the two loci. Eight plants were identified with recombinations between lin and MtN6, thus demonstrating that lin and MtN6 are distinct loci. MtN6 was determined to map north of lin on LG1 to a position most closely linked to the genetic marker DK369R.
The morphological phenotype of lin suggested that LIN is necessary for nodule differentiation. To determine the nature of the developmental block in lin, we used RNA blots to examine the expression of four nodulin genes as markers of various stages of nodulation. The early nodulins RIP1 and ENOD40 were selected as markers of early responses to Nod factor. As shown in Figure 4, the onset of ENOD40 induction was similar between wild-type and mutant plants, with increased transcript abundance evident at 1 dpi. RIP1 was also induced in lin and wild-type plants, with transcript first detectable in both genotypes at 6 h postinoculation. ENOD2 and ENOD8 were chosen as markers of nodule differentiation. Observations on RNA blots revealed that, in wild-type plants, ENOD2 expression commenced about 4 dpi, and ENOD8 transcript accumulated by 6 dpi. Transcripts corresponding to these two nodulins could not be detected in lin (Fig. 4). A duplicate blot, with RNA from independent biological replicates, verified the expression patterns of these nodulins (data not shown).
Together, these results indicated that the early responses of the plant to rhizobia were present in lin. By contrast, cytological events related to nodule organogenesis, including nodule differentiation, were partially or completely blocked in lin. We therefore chose three additional markers of early primordium formation and infection thread penetration (ENOD20, MtN6, and CCS52A) to examine the molecular phenotype of lin. Real-time PCR analysis indicated that both genotypes showed an increasing expression of ENOD20 in inoculated roots through 5 dpi (Fig. 5A), with slightly higher expression in wild type relative to lin at 5 dpi. By contrast, expression of MtN6 in lin was significantly lower at all time points compared to wild type. MtN6 induction in lin was 1.5-fold at 1 dpi and reached a maximum of 2.5-fold at 18 dpi (Fig. 5B), as compared to the wild-type roots in which a dramatic increase of MtN6 occurred between 1 dpi (2.7-fold) and 10 dpi (380-fold). For both genes, the differences in basal levels of expression in roots between the two genotypes were slight, and transcript abundance in uninoculated roots varied little over time.
During the maturation of indeterminate nodules, selected cells within the primordium switch from mitotic cycles into endoreduplicating cycles. This is mediated by the cell cycle-switching gene CCS52A. We analyzed the expression of CCS52A in lin compared to that of wild-type plants. As shown in Figure 5C, transcripts increased in both wild-type and lin genotypes, although the response was both earlier and of substantially greater magnitude in wild type compared to lin (i.e. 50- to 500-fold greater induction in wild type).
A Symbiotic Mutant, lin, Identifies a Gene in M. truncatula Required for Persistence of Rhizobial Infections and Differentiation of Nitrogen-Fixing Nodules In an effort to identify plant genes required for infection maintenance and nodule organogenesis in the legume-Rhizobium symbiosis, we identified a symbiotic mutant in M. truncatula called lin that is blocked in development prior to invasion of nodule primordia by infection threads. In lin, the number of infections is reduced by 5-fold compared to wild type. Infections that initiate in lin are of abnormal structure and are arrested within the root epidermis, failing to penetrate cells of the root cortex. Genetic analysis of lin showed that the mutation is conferred by a single recessive allele, and genetic mapping has placed LIN in the lower arm of LG1.
The lin phenotype supports the hypothesis that progressive invasion of the infection thread is necessary for complete nodule organogenesis (Dénarié et al., 1992 The lin mutation blocks the penetration of the infection thread into the cortex but permits mitotic activation of cortical cells and the initial stages of primordium formation. The lin phenotype could be due to one or more factors. Failure to transduce the Nod factor or other signals properly could result in lack of continued symbiotic development. Conversely, deployment of an inappropriate defense response could result in an active infection arrest. The former hypothesis was addressed in further phenotypic characterization of the mutant.
In M. truncatula, the host plant regulates the number of persistent rhizobial infections and nodules by at least two distinct mechanisms. Ethylene insensitivity, as caused by the skl mutation (Penmetsa and Cook, 1997
While recent successes in map-based cloning have advanced understanding of plant genetic control of Nod factor perception, knowledge of genes regulating stages of nodule development and infection is still rudimentary. This intermediate stage encompasses a range of activities, including cortical cell activation leading to formation of primordia, infection thread formation and penetration into the primordium, release of bacteria as membrane-bound organelles within cells of the primordium, and endoreduplication and subsequent differentiation of these cells. In comprehensive genetic and phenotypic analyses of pea (Pisum sativum) mutants, Tsyganov et al. (2002)
We used northern-blot and real-time PCR analysis to analyze expression of various nodulin genes according to the lin mutation, and by comparison to wild type we were able to infer additional aspects of the lin phenotype. As depicted in Table III, the composite of the current results with those from previous studies allows the ordering of both gene expression and the action of genetic loci within the context of a pathway for nodule development and gene transcription. Three genes (RIP1, ENOD20, and ENOD40) were induced in inoculated lin roots at levels nearly comparable to wild type. The induction of RIP1 and ENOD40 is consistent with the ability to perceive and transduce the Nod factor signal by lin, as in wild type (Crespi et al., 1994
The M. truncatula mutants hcl, nsp1, and nsp2 also show modest-to-normal induction of one or more early nodulins that are induced in response to Nod factor, but otherwise have different phenotypes from lin (Catoira et al., 2000 , 2001; Oldroyd and Long, 2003). Mutants at these three loci exhibit root hair deformation, but do not become infected. Also, cell divisions are induced in the inner root cortex of hcl by rhizobia, but do not persist to generate a nodule primordium. We hypothesized that a class of molecular markers for infection initiation and primordium formation would differentiate lin from the other two mutants. The cell cycle switch gene CCS52A is a suitable marker for the transitional stages between early Nod factor perception and nodule differentiation. CCS52A was induced around the same time in lin and wild type, although its transcript levels were much lower in the mutant. This suggests that LIN is required for full expression of CCS52A, but not for its induction in response to Rhizobium. Reduced expression of CCS52A in lin could indicate that lin nodules undergo only a modest increase in ploidy. The infection arrest phenotype of lin supports the prediction from Vinardell et al. (2003) that posits that ramification of infection threads is necessary for full activation of CCS52A. In addition to results from monitoring CCS52A expression presented here, a more in-depth search for genes involved in primordium formation is currently under way using comparative cDNA microarray analysis of lin and wild type. A similar approach, based on identification of genes that are misregulated in lin, should also identify genes that function in primordium differentiation and infection thread growth.
Three genes (MtN6, ENOD8, and ENOD2) were identified that fail to be induced in lin. Consistent with previous findings, levels of MtN6 increased in wild-type plants during infection progression and nodule development. In contrast, lin roots showed minimal induction of MtN6, suggesting that preinfection events related to primordium differentiation are not activated in lin. The similarity of the lin-arrested infections and nodule structures to those of empty alfalfa nodules induced by Rhizobium meliloti EPSI (exo–) mutants (Yang et al., 1992 In summary, the phenotypic analyses of lin presented here suggest that LIN functions downstream of initial events in Nod factor signal transduction, but is required for both infection persistence and nodule differentiation. We hypothesize that LIN may also be required to suppress a host defense response, allowing some infections to proceed. Further work to investigate this hypothesis is in progress. The map-based cloning and molecular characterization of LIN, initiated here with genetic mapping of the lin mutation to LG1 of M. truncatula, should provide further insight into LIN function.
Plant Material and Growth Conditions
Medicago truncatula cv Jemalong genotype A17 was used as the wild-type control for phenotypic and genotypic analysis. The plants were grown as previously described by Cook et al. (1995)
To visualize infection of roots and nodules by rhizobia, tissues inoculated with the rhizobia bearing the lacZ construct were stained for
To facilitate the examination of the internal structure of the root, segments containing infections were dissected from the main root, dehydrated in an ethanol series, and embedded in LR White acrylic resin as described previously (Sherrier and VandenBosch, 1994
Nitrogenase activity of inoculated roots was determined by assaying acetylene reduction, using roots harvested from an aeroponic chamber 21 dpi (Somasegaran and Hoben, 1994
For allelism tests of lin with other nodulation mutants, either pollen from plants homozygous for other nodulation mutants was applied to the stigmatic surface of the male-sterile line of lin to obtain F1 seeds, or vice versa. The nodulation phenotype of the F1 progeny for each cross was assessed to determine whether the mutant was allelic to lin.
For mapping studies, a mapping population was constructed by crossing lin in the tap genetic background (that does not affect nodulation) with ecotype A20. F1 seeds were collected and seedlings were tested for nodulation phenotype, as described above. F2 seeds were obtained by self-pollinating the F1 seeds. A population of 125 F2 individuals was visually screened twice for the presence or absence of nodules at 10 and 30 dpi. A trifoliate leaf from each of the F2 plants was used to extract genomic DNA for PCR as described (Williams and Ronald, 1994
M. truncatula plants were germinated, transferred to an aeroponic chamber, grown in nitrogen-free media for 5 d, and inoculated with S. meliloti strain ABS7M. At various times before or after inoculation, plants were harvested and the bottom 2 cm of the root tip were removed to eliminate the root meristem tissue, as in Peng et al. (1996)
Root tissues were collected from infected plants at 1, 3, 5, 10, and 18 dpi and also from uninoculated, uninfected plants of equivalent age. For harvests at early time points, the root tips were excised from the rest of the root before freezing samples for RNA extraction. In contrast, for plants grown for 10 and 18 dpi and from their equivalent uninfected plants, approximately 10 cm of the root tissues targeting the nodulation competent zone were harvested. Total RNA was extracted using the RNeasy plant kits (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA samples were harvested for each time point from three independent biological replicates that were then pooled in equal amounts to average out biological variability. For cDNA synthesis, DNase-treated total RNA (Ambion, Austin, TX) was used and the reverse transcription was carried out with the oligo(dT) primers (Invitrogen, Carlsbad, CA) using the C. Therm. polymerase reverse transcription kit (Roche Applied Sciences, Indianapolis) following the manufacturer's directions. Gene-specific primers were designed based on the sequence information available at NCBI (http://www.ncbi.nlm.nih.gov) and the Institute for Genomic Research MtGI, version 7.0 (http://www.tigr.org/tdb/tgi/mtgi) databases. To allow detection of amplification from contaminant genomic DNA, primers were located within exons and selected to span one or more introns. The specific primers were RT1F (5'-TTGGCTGTTGGTTTGGGTAAC-3') and RT1R (5'-TACTCATCTTGCTGTTGGAAC-3') for CCS52A (TC37234; GenBank accession no. AF134835); RT2F (5'-CAAATGGAACTTCTTGGACTC-3') and RT2R (5'-CCAACTATCTCACCAGCAACT-3') for MtN6 (TC88012; GenBank accession no. AJ133118); RT3F (5'-CTCTTATTCCACATCTATC-3') and RT3R (5'-ACCCGTTG GGCCTCTAACT-3') for ENOD20 (TC77148; GenBank accession no. X99467). Transcripts detected from two housekeeping genes (actin [TC85697] and an O-linked N-acetyl glucosamine transferase [TC77416], otherwise called secret agent [Hartweck et al., 2002 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AJ133118, AF134835, and X99467.
We thank Thierry Huguet and Pascal Gamas for sharing information on the map location of MtN6 prior to publication. We gratefully acknowledge Jeff Esch, Michelle Graham, and M. David Marks for their technical assistance in the real-time PCR analysis. We also thank Lynn Hartweck for sharing information on secret agent as a constitutively expressed gene prior to publication. Received May 3, 2004; returned for revision July 26, 2004; accepted August 7, 2004.
1 This work was supported by a grant from the U.S. Department of Agriculture-National Research Initiative (award no. 98–35305–6686 to K.VdB.), by funding from the University of Minnesota (to K.VdB.), and by funding from the University of North Texas (to R.D.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.045575. * Corresponding author; e-mail: kvandenb{at}cbs.umn.edu; fax 612–625–1738.
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RESULTS
-galactosidase activity. B and C, Segments of lin root showing the recurrent occurrence of primordia (arrowheads in B) and infections (arrows in C) at 18 dpi. D and F, Wild-type infections. A root harvested at 1 dpi (in D) shows an infection thread (arrow) penetrating through the root hair and into the cortical cell layer, while F depicts a successful infection (arrow) ramifying within a developing primordium (np) at 3 dpi. E and G, Infections in the lin mutant. E, Infection (arrow) arrested within the root hair at 1 dpi. G, Arrested infection (arrow) that fails to penetrate the nodule primordium (np) at 3 dpi. Bars = 50 µm.
