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First published online September 23, 2009; 10.1104/pp.109.143933 Plant Physiology 151:1239-1249 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
LIN, a Novel Type of U-Box/WD40 Protein, Controls Early Infection by Rhizobia in Legumes1,[C],[W],[OA]Institute of Genetics, Biological Research Center, Szeged 6726, Hungary (E.K., B.O., A.B., A.L., K.K., G.E.); and Department of Disease and Stress Biology (P.K., M.M., P.M., G.E.D.O.) and Department of Molecular Microbiology (A.B.H., J.A.D.), John Innes Centre, Norwich NR4 7UH, United Kingdom
The formation of a nitrogen-fixing nodule requires the coordinated development of rhizobial colonization and nodule organogenesis. Based on its mutant phenotype, lumpy infections (lin), LIN functions at an early stage of the rhizobial symbiotic process, required for both infection thread growth in root hair cells and the further development of nodule primordia. We show that spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase is independent of LIN; thus, LIN is not necessary for nodule organogenesis. From this, we infer that LIN predominantly functions during rhizobial colonization and that the abortion of this process in lin mutants leads to a suppression of nodule development. Here, we identify the LIN gene in Medicago truncatula and Lotus japonicus, showing that it codes for a predicted E3 ubiquitin ligase containing a highly conserved U-box and WD40 repeat domains. Ubiquitin-mediated protein degradation is a universal mechanism to regulate many biological processes by eliminating rate-limiting enzymes and key components such as transcription factors. We propose that LIN is a regulator of the component(s) of the nodulation factor signal transduction pathway and that its function is required for correct temporal and spatial activity of the target protein(s).
The soil bacteria rhizobia are able to establish nitrogen-fixing symbioses with leguminous plants by inducing the formation of a new organ, the nodule, on the roots of the host plant. Symbiotic infection is initiated and maintained by an exchange of signaling molecules between the host plant and the microsymbionts. In most legumes, flavonoid compounds produced by leguminous plants activate bacterial regulators to induce nodulation (nod) genes required for the synthesis of lipochitooligosaccharide Nod factors. Nod factors initiate multiple early responses on plant hosts, including a burst of intracellular calcium levels in root hairs, calcium oscillations (calcium spiking), and the induction of nodulation-specific genes whose products are referred to as "nodulins." In addition, they induce a rearrangement of the root hair cytoskeleton, leading to root hair deformation and curling, which traps surface-attached rhizobia, establishing a site that acts as an infection focus. Bacteria penetrate into the curled root hairs toward the root cortex via host-derived tubular structures called infection threads. Simultaneously, Nod factors stimulate the reinitiation of mitosis in cortical cells, leading to the formation of nodule primordia, which give rise to the cells that receive the invading bacteria (Oldroyd and Downie, 2008  ). Exopolysaccharides, capsular polysaccharides (K antigens), and lipopolysaccharides serve as further bacterial signals required for successful infection of nodules in many legumes (Jones et al., 2007). The infecting rhizobia are released into intracellular membrane compartments of plant origin called symbiosomes, where they differentiate into bacteroids capable of reducing atmospheric nitrogen to ammonium, which is provided to the plants in exchange for carbon and amino acid compounds (Brewin, 2004).
Analysis of nodulation-defective Medicago truncatula, Lotus japonicus, and pea (Pisum sativum) mutants has led to insights into the mechanisms by which Nod factors are perceived and trigger subsequent signal transduction cascades (Geurts et al., 2005
The lumpy infections (lin) mutant (line C88) was identified as a M. truncatula mutant, in which infection was arrested and reduced to one-quarter of the frequency seen in wild-type plants (Penmetsa and Cook, 2000 In this work, we show the identification of the LIN gene, revealing that LIN encodes a large protein containing multiple domains including a U-box (modified RING) domain, indicating a function as an E3 ubiquitin ligase. Spatiotemporal analysis of the promoter activity of LIN demonstrated that the expression of the gene correlated with the early nodule primordia formation and bacterial invasion during the symbiosis. We used a gain-of-function mutation in CCaMK that induces spontaneous nodulation in the absence of rhizobia to show that LIN is not required for nodule organogenesis. This indicates that LIN functions exclusively during rhizobial colonization and that the defect in nodule development in lin is a response to aborted infection.
LIN Is Not Required for Nodule Organogenesis Ethyl methanesulfonate (EMS) and fast-neutron-mutagenized M. truncatula populations were screened to identify new loci required for nodulation; two of the mutants identified had phenotypes similar to lin, namely impaired nodulation and infection with the infection threads arrested in the root hair cells. Although nodule primordia emerged 3 to 4 d after inoculating the roots with wild-type rhizobia, nodule development was always blocked before the stage of nodule differentiation. No nonsymbiotic phenotype was detected in these mutants. Genetic crosses revealed that the two mutants (EMS6:T7 and 14P) carried mutations allelic to lin-1 (Table I) but not allelic to the other nodulation mutations tested (data not shown). EMS6:T7 was derived from EMS mutagenesis and will be designated as lin-2, while 14P was derived from fast-neutron mutagenesis and will be designated as lin-3.
To determine if LIN is required for nodule organogenesis, we tested whether an autoactive form of CCaMK could induce spontaneous nodulation in lin mutants. In M. truncatula, transgenic expression of an autoactive CCaMK (DMI31–311), composed of the kinase domain alone, is capable of inducing spontaneous nodules and has been useful in determining the order of gene product function within the early Nod factor signaling pathway relative to CCaMK (Gleason et al., 2006 ). When we transformed lin-1, lin-2, and lin-3 mutants with this autoactive CCaMK construct, we could clearly detect nodules on the lin-1 (three of 21 plants), lin-2 (nine of 19 plants), and lin-3 (40 of 103 plants) mutants. The observation that spontaneous nodules were induced on 52 of 143 lin mutant plants transformed with autoactive CCaMK suggests that LIN is not essential for nodule organogenesis; therefore, the defects in nodule development observed in the lin mutants during rhizobial invasion are likely to be a result of the abortion of bacterial infection.
Preliminary mapping data previously positioned lin-1 (C88) between molecular markers DSI and SCP on the lower arm of linkage group 1 (LG1) within a relatively large distance of several centimorgan (Kuppusamy et al., 2004
Further sequencing of this region in lin-1 and comparisons with the sequence from the wild type revealed a single nucleotide difference in lin-1 in the predicted coding region MTCON310-47 (indicated by the star in Fig. 1B). Since no EST had been reported for this gene, we validated that this region was actively transcribed in M. truncatula. PCR amplification products could easily be obtained from plant cDNA isolated from roots 4 d after Sinorhizobium meliloti inoculation. The cDNA of MTCON310-47 could be assembled from the sequences of overlapping fragments. The intron/exon boundaries were similar to what had previously been predicted for the genomic sequence, with the exception that exon 12 was found to be 24 bp longer (indicated by a white triangle in Fig. 1C), resulting in a predicted total protein of 1,488 amino acids. The mutation we identified in lin-1 was in the last nucleotide position of intron 4 (indicated by an arrow in Fig. 1C). Reverse transcription (RT)-PCR amplification of the affected region using primers Lin-3F (in exon 3) and Lin-3R (in exon 5) on lin-1 RNA samples of inoculated roots revealed that there was indeed an error in the RNA splicing, resulting in a longer transcript (Fig. 2A). Sequencing of this longer fragment showed that due to the mutation, intron 4 was not spliced out during mRNA processing (while correct splicing of intron 3 was detected in the same fragment), causing a premature stop codon to be introduced. Amplification of all other parts of the lin-1 cDNA resulted in the expected fragment sizes (e.g. the fragment amplified by Lin-8R and Lin-8R primers in Fig. 2A).
Sequencing of the genomic DNA amplified from the lin-2 mutant revealed a point mutation in the first exon that appears at nucleotide position 662 in the cDNA sequence, introducing a premature stop codon into MTCON310-47 very early in this gene (indicated by an arrow in Fig. 1C). In addition, Southern analysis of lin-3 revealed an apparent large deletion or rearrangement in this same gene (Fig. 2B). RT-PCR experiments on lin-3 demonstrated the presence of a short mRNA (1,568 nucleotides) transcribed from this allele (indicated by an arrow in Fig. 1C). Another mutant line, C105, originating from the same population (Penmetsa and Cook, 2000 ) and having a similar phenotype to C88 (lin-1), is allelic with C88 (R. Dickstein, personal communication); sequencing of MTCON310-47 in C105 revealed the same mutation, suggesting that C88 and C105 are siblings. The identification of mutation events in three different alleles of lin provides strong evidence that MTCON310-47 is indeed LIN.
To confirm that MTCON310-47 corresponds to LIN, we tested complementation of the lin alleles with a construct carrying MTCON310-47 expressed off the 35S promoter and also harboring a constitutively expressed GFP reporter gene to facilitate the identification of the transgenic roots. The lin mutants were transformed using Agrobacterium rhizogenes-mediated hairy root transformation, and transgenic hairy roots were detected by the fluorescent GFP signal (Fig. 3A). For screening the symbiotic phenotype of the roots, wild-type symbiotic bacteria were applied on the well-grown root systems following 1 week of nitrogen starvation. Complementation of the symbiotic phenotype of all three lin mutant alleles was observed, indicated by the formation of mature nodules induced by S. meliloti strain 1021 carrying a hemA promoter:lacZ fusion (Fig. 3B). The lin mutant alleles do form bumps following S. meliloti inoculation; however, in these complementation experiments, we could clearly discriminate between the mature complemented nodules that were infected (Fig. 3B) and the nodule primordia bumps, where infections always arrested in the root hairs on the nontransgenic roots of lin mutants or on transgenic roots induced by A. rhizogenes carrying the empty vector (Fig. 3C). Using β-galactosidase staining of the symbiotic bacteria, we could clearly see the presence of rhizobia within the nodule and in infection threads ramifying throughout the nodules on complemented roots. The identification of mutations in MTCON310-47 in lin-1, lin-2, and lin-3, coupled with the complementation of these mutants by this gene, proves that MTCON310-47 encodes LIN.
Identification and Transcomplementation of a L. japonicus lin Mutant
Mutants of L. japonicus with a phenotype similar to the M. truncatula lin mutant were described previously, and two of the mutant loci, sym7 and itd3, map to a 10-centimorgan region on the upper arm of chromosome 5 (Lombardo et al., 2006
The MtLIN-like gene was amplified and sequenced from mutants SL1450-5 (carrying an allele of sym7) and SL1947-2 (carrying itd3), revealing one change, a G-to-A substitution at position 3,799, in SL1450-5 and no change in SL1947-2. This mutation in SL1450-5 resulted in the alteration of Asp-1267 (GAC) to Asn (AAC). Roots of SL1450-5 were transformed with the same MtLIN construct that was used for complementation of the M. truncatula lin mutants. MtLIN complemented both nodulation (Table II
; Fig. 4A)
and infection (Fig. 4B) of SL1450-5 in transformed hairy roots. Normal-looking pink nodules could be observed in the complemented plants, indicating that they were functional; nodulation, scored only using those transgenic roots showing GFP fluorescence, was significantly different from the controls lacking MtLIN (Table II). This shows that (1) the L. japonicus mutant phenotype can be rescued by the M. truncatula wild-type LIN gene, (2) MtLIN is the ortholog of LjSYM7, and (3) the identified mutation caused the nodulation-defective phenotype of SL1450-5. A few nodules were observed on hairy roots in the negative controls (Table II), but microscopy revealed only a few infected cells in these nodules, as had been seen previously with the SL1450-5 mutant (Lombardo et al., 2006
LIN Belongs to a Unique Family of Proteins Containing Domains with Homology to E3 Ubiquitin Ligases Analysis of LIN revealed regions with high similarity to protein domains of known function. A U-box domain is present between residues 516 and 580 (Fig. 5). U-box domains are modified RING finger domains without the full complement of Zn2+-binding ligands and known for their E3 ubiquitin ligase activity. At the C-terminal region of the protein, the National Center for Biotechnology Information (NCBI) conserved domain search program identified a large region belonging to the WD40 superfamily (Fig. 5), while the predictions by InterProScan suggested three clear WD40 repeats with scores above the threshold from several programs (SMART, Pfam, SPRINT). According to the PANTHER classification system (www.pantherdb.org/panther/), this part shows homology to a more general family of F-box and WD40 domain proteins (PTHR22844) trained by 71 sequences from several organisms. Between these two main identified domains, a large Armadillo repeat-type region was predicted (Fig. 5) by only one protein analysis program (residues 662–1,001; InterPro-Gene3D: IPR011989 Armadillo-like helical domain). No further significant similarity to known domains was found. By comparing the predicted domain structure of LIN and the identified cDNA sequences of the three lin alleles in M. truncatula, it is clear that none of the possible truncated proteins would carry the U-box, Armadillo-like, and WD40 domains. The mutation identified in L. japonicus SL1450-5 mutant altered Asp-1267 (GAC) to Asn (AAC) in the second predicted WD40 repeat, indicating that the WD40 motif is essential for function.
When the Conserved Domain Architecture Retrieval Tool was used to find proteins with similar structural features, the closest group was proteins with the combination of RING finger and WD40 repeat domains (ring finger and WD40 repeat domain 3 Homo sapiens). Another set of proteins with a similar structural arrangement involves pre-mRNA processing factor 19 homologs (Bos taurus), which contain U-box and WD40 repeats, but always together with a PRP19/PSO4 domain. An additional large family includes proteins with the combination of F-box/WD40 repeat domains (like Pop1 from Schizosaccharomyces pombe). However, LIN is unusual when comparing with any of these families, as it contains a U-box instead of an F-box or RING domain and is exceptionally large with significant regions of unknown function. The structure of LIN (a large and novel N-terminal region, followed by a U-box, a probable Armadillo repeat, and WD40 repeats at the C-terminal domain) appears to be unique for plants, since genes with similar structure could not be found in other organisms. Genes homologous to LIN are clearly present in poplar (Populus trichocarpa) and grapevine (Vitis vinifera), identified as being duplicated in the Populus genome (Pt LGII, accession no. NC008468; Pt LGV, accession no. NC008471). The hypothetical protein product of the Vitis homolog is predicted under the GenBank accession number CAN74785. The amino acids of the predicted proteins are closely aligned, even the N-terminal region between residues 1 and 450 shows strong similarity with remarkably high conservation at positions 1 to 300 (Fig. 5; Supplemental Fig. S1). The next most similar protein to LIN was identified in rice (Oryza sativa; Os01g0229700; Fig. 5; Supplemental Fig. S2) and contains similar domain structure, but the level of similarity clearly drops at the N-terminal region, indicating that domain to be diverse. In the Arabidopsis (Arabidopsis thaliana) genome, no single homologous sequence could be identified, and no gene encoding a protein with similar domain structure was identified. Instead, two proteins show homologous regions with parts of the LIN protein: At3G06880 codes for a longer protein with WD40 repeats, while At1G23030 codes for a shorter U-box-containing protein (Fig. 5; Supplemental Figs. S3 and S4).
In order to reveal the promoter activity of the M. truncatula LIN gene throughout the nodulation process, a 1.2-kb segment upstream of the coding region of LIN was fused to the GUS reporter gene. This pLIN-GUS construct was subsequently introduced into M. truncatula A17 plants by A. rhizogenes-mediated transformation. Transformed hairy roots were monitored for GUS activity on uninfected roots and at different time points after inoculation with S. meliloti. There was no detectable signal of GUS activity throughout most parts of the uninoculated roots, except a very faint signal just above the detection limit at the apical region (Fig. 6A). Three days after inoculation with rhizobia, GUS activity seemed to be associated with dividing cortical cells, leading to the formation of nodule primordia (Fig. 6B). Six days after inoculation, strong overall GUS staining was detected in the young, emerging nodules, where infection of plant cells by rhizobia takes place (Fig. 6C). In elongated mature nodules (21 d after inoculation), strong GUS activity was detected but was mainly restricted to a relatively broad area of nodule apices including the infection zone. Much lower expression was detected in the nitrogen-fixing zone (Fig. 6D).
These data indicated that the cloned promoter segment enabled a gene expression associated with the bacterial infection as well as with the cortical cell division, leading to the formation of symbiotic nodules. Although this showed an activity that strongly correlated to the position where the expression of the LIN gene would be needed to recondition the mutant phenotype (i.e. early nodule primordia), to further support its activity, a complementation experiment was carried out using the wild-type LIN cDNA driven by the same promoter segment. Fully developed nodules appeared on the transgenic roots of lin-1 mutant plants, and bacterial invasion was detected in the apical zone of the nodules (Supplemental Fig. S5). This confirmed that promoter activity of this segment ensures a LIN expression necessary for nodule development and restoration of the infection process during early nodule invasion, but not sufficient for the complete occupation of the nodule by bacteria. This suggests that the function of LIN might also be needed at later steps of nodule invasion and occupation, and further regulatory elements in a larger promoter segment in the 5' upstream region of LIN would be responsible for its expression and function at these later stages.
The formation of a nodule requires the coordinated development of rhizobial colonization and nodule organogenesis. These two processes are coordinated both spatially and temporally to ensure rhizobial infection of the developing nodule. However, rhizobial infection and nodule organogenesis can be separated genetically (Gleason et al., 2006 ; Murray et al., 2006; Tirichine et al., 2006a, 2006b), indicating that these two processes constitute different developmental pathways. Based on its mutant phenotype, LIN functions at an early stage of the rhizobial symbiotic process, required for both infection thread growth in root hair cells and the further development of nodule primordia. The lin mutation in L. japonicus SL1450-5 did not affect Nod factor-induced calcium spiking (Lombardo et al., 2006). No apparent nonsymbiotic phenotypes were observed in any of the three M. truncatula lin mutants, and colonization of the lin-1 mutant by mycorrhizal fungi is similar to that of wild-type M. truncatula (M. Harrison, personal communication). Gain-of-function mutations in CCaMK (autoactive CCaMK) allowed nodule development in the absence of rhizobial infection, and the use of this mutant revealed that LIN is not necessary for nodule organogenesis. From this, we infer that LIN predominantly functions during rhizobial colonization and that the abortion of this process in lin leads to a suppression of nodule development.
Detailed genetic studies in M. truncatula, L. japonicus, and pea have revealed an ordered array of loci functioning at different stages of nodulation (Tsyganov et al., 2002
The LIN protein contains many domains in which the presence of a U-box places it among E3 ubiquitin ligases, while other regions might be responsible for different interactions with target/substrate proteins or regulation of LIN by posttranslational modifications. E3 ubiquitin ligases play roles in the ubiquitination of the target protein achieved by enzymatic reactions that act in concert. The substrate proteins are labeled with ubiquitin, which serves as a degradation tag, in three consecutive steps catalyzed by the enzymes E1, E2, and E3, leading to proteolysis of target proteins by the 26S proteasome complex (Glickman and Ciechanover, 2002 The complete structure of LIN appears to be unique to plants, since genes coding for proteins with similar structures could not be found in other organisms. Even in the plant kingdom, it is sometimes absent (e.g. in the Arabidopsis genome, no single homologous sequence could be identified). Instead, two proteins show homologous regions with parts of the LIN protein: At3G06880 codes for a longer protein with WD40 repeats, while At1G23030 codes for a shorter U-box-containing protein. On the other hand, the genome of the phylogenetically more distant rice does carry a gene coding for a protein with a domain structure (Os01g0229700) similar to LIN, although the level of similarity is poor at the N terminus. L. japonicus, P. trichocarpa, and V. vinifera show genes with a high degree of similarity to LIN. We show that the homologous protein in L. japonicus has an equivalent function to LIN, although the presence of homologs in nonlegumes suggests functions unrelated to nodulation in these species.
The emerging picture for ubiquitin regulation in plant-microbe interactions is supported so far mostly by examples in plant defense, suggesting multiple levels of regulation: from the resistance proteins to downstream signaling components and regulators (Zeng et al., 2006
There are already indications for an involvement of E3 ligases in the legume/rhizobia symbiosis, with two E3 ligases showing a role in nodule development. Recently, nsRING, a novel RING finger protein required for rhizobial invasion and nodule formation, was identified in L. japonicus (Shimomura et al., 2006
Plant Growth and Bacterial Strains
Medicago truncatula ‘Jemalong’ genotype A17 was used as the wild-type control for phenotypic and genotypic analyses. The plants were grown as described previously by Cook et al. (1995)
Mapping was done on an F2 segregating population originating from a cross between C88 and A17 as described by Kuppusamy et al. (2004)
The following primer pairs were used to demonstrate the imperfect splicing of the mRNA in the lin-1 mutant: Lin-3F, 5'-GGATGAAGATGTTGAACCAA-3', and Lin-3R, 5'-CCTGTGATTGGACAAACAA-3'; Lin-8F, 5'-GGACGCAAGGAAGAGAAT-3', and Lin-8R, 5'-CCAGTGAAGAATGAAGTTGATG-3'. Primers Lin-3Fb (5'-TTGTTTGTCCAATCACAGG-3') and Lin-12Rc (5'-ACAGTCCTGCAAGTTTTCTGATGT-3') were used to amplify fragments for the Southern hybridization. All amplification reactions were carried out as described earlier (Endre et al., 2002a
LIN sequences and homologs were aligned using VectorNTI. For different domain, motif, and structure predictions, the following programs/Web sites were used: NCBI conserved domain search program on A Conserved Domain Database and Search Service, version 2.16; Conserved Domain Architecture Retrieval Tool (searches the NCBI Entrez Protein Database for similarity based on domain architecture, defined as the sequential order of conserved domains in proteins; Geer et al., 2002
For complementation experiments, LIN cDNA was recombined from the pCR8GW-TOPO entry clone into the pK7WG2D vector (Karimi et al., 2002
To generate the pLIN-GUS fusion construct, a 1.2-kb segment upstream the coding region of LIN was amplified and cloned into the binary vector pMDC164 (Curtis and Grossniklaus, 2003 Sequence data from this article are deposited in the GenBank/EMBL data libraries under the following accession numbers: MtLIN, EU926660; MtLIN_CDS, EU926661; Mtlin-1, EU926662; Mtlin-2, EU926663; Ljlin_CDS, EU926664; and Ljlin-1, EU926665.
The following materials are available in the online version of this article.
We greatly appreciate the technical assistance of Sandor Jenei, Andrea Toth, Zsuzsa Liptay, and Erika Veres. We thank Kavitha Kuppusamy and Kathryn A. VandenBosch for the F2 seeds of the M. truncatula lin-1 segregating population. Received June 30, 2009; accepted September 18, 2009; published September 23, 2009.
1 This work was supported by the Hungarian Scientific Research Fund (grant nos. OTKA T046819, D048451, and K76843); by the National Research and Development Program (grant no. NKFP 4/031/2004), the Economic Competitiveness Operative Programs (grant no. GVOP–3.1.1–2004–05–0101/3.0), and the Biotechnology and Biological Research Council in the United Kingdom via a grant in aid and grant no. BB/D521749/1; by the European Union (grant nos. RTN–CT–2003–505227 and MRTN–CT–2006–035546); and by Janos Bolyai postdoctoral fellowships to E.K. and G.E. 
2 Present address: Agricultural Biotechnology Centre, Gödöllö 2100, Hungary.
3 Present address: Department of Molecular Biology, University of Aarhus, 8000 Aarhus C, Denmark. 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: Gabriella Endre (endre{at}brc.hu).
[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.109.143933 * Corresponding author; e-mail endre{at}brc.hu.
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2 test).
(Gibco BRL) and One Shot Omnimax 2T1 (Invitrogen) strains were used. S. meliloti and E. coli strains were grown at 30°C and 37°C, respectively, in Luria-Bertani medium supplemented with antibiotics when required. In hairy root transformation experiments, Agrobacterium rhizogenes ARqua1 strains electroporated with the appropriate binary vector constructs were used.