Legume pectate lyase required for root infection by rhizobia

Edited by Eva Kondorosi, Institute for Plant Genomics, Human Biotechnology and Bioenergy, Szeged, Hungary, and approved November 14, 2011 (received for review August 30, 2011)
December 27, 2011
109 (2) 633-638

Abstract

To allow rhizobial infection of legume roots, plant cell walls must be locally degraded for plant-made infection threads (ITs) to be formed. Here we identify a Lotus japonicus nodulation pectate lyase gene (LjNPL), which is induced in roots and root hairs by rhizobial nodulation (Nod) factors via activation of the nodulation signaling pathway and the NIN transcription factor. Two Ljnpl mutants produced uninfected nodules and most infections arrested as infection foci in root hairs or roots. The few partially infected nodules that did form contained large abnormal infections. The purified LjNPL protein had pectate lyase activity, demonstrating that this activity is required for rhizobia to penetrate the cell wall and initiate formation of plant-made infection threads. Therefore, we conclude that legume-determined degradation of plant cell walls is required for root infection during initiation of the symbiotic interaction between rhizobia and legumes.
The infection of legumes by nitrogen-fixing rhizobia occurs via plant-made infection threads (ITs). These tube-like structures, lined with a plant cell wall and membrane, are usually initiated in curled root hairs and grow down through the root hair and continue growing through epidermal and cortical cells (1). When the growing IT reaches the dividing root cells that make up the nodule primordium, the plant cell wall of the IT is lost and the bacteria are budded off into the plant cytoplasm surrounded by a plant-derived membrane. The bacteria then differentiate into nitrogen-fixing forms called bacteroids and in the mature nodule, they fix N2, producing ammonia that is translocated to the plant.
The initiation and growth of ITs require signaling between rhizobia and legumes. Rhizobial nodulation (Nod) factors activate nuclear-associated calcium spiking via a signaling cascade that requires LysM-receptor kinases and a leucine-rich repeat receptor-like kinase in the plasma membrane and nucleoporins and ion channels in the nuclear membrane. The subsequent activation of a calcium and calmodulin-dependent kinase then activates transcription factors required for the induction of nodulation and infection genes (2). Nod factors also induce a calcium influx that is associated with depolarization of the plasma membrane; this calcium influx has been proposed to be important for initiation of infection (3). Oligosaccharides derived from the synthesis of the rhizobial exopolysaccharide also play a crucial role in initiation of infection, possibly by suppressing plant defense responses (4, 5). Membrane-associated remorins and flotillins that promote protein interactions and alter membrane dynamics are also important for infection (6, 7).
Initiation of infection in root hairs requires localized degradation of the root-hair cell wall and the initiation of inward growth of the cell wall and membrane. Genes that play a role in remodeling the cytoskeleton are required for infection initiation (8, 9). However, although other genes with both identified (1014) and undefined roles (1) have been characterized, these genes have not yet given insights into the mechanistic changes required for initiation of root-hair infection.
It has been recognized for over 120 y that local penetration of the plant cell wall is required for legume infection (15) and there are two schools of thought as to how this penetration is achieved. Bacterially produced enzymes have been proposed to locally degrade the root-hair cell wall (16, 17). Alternatively, plant cell-wall degrading enzymes induced in response to rhizobia may be responsible (1820) and indeed Nod factors can promote localized cell-wall degradation (21). However, there is no unequivocal evidence as to which of these two models is correct. In this work we demonstrate that rhizobia induce a Lotus japonicus pectate lyase, which is required for root-hair and nodule infection by rhizobia.

Results

Identification of an Infection Mutant of L. japonicus.

A L. japonicus mutant (SL5711-2) defective for infection by Mesorhizobium loti was identified and was of interest because: (i) unlike WT, most infections were arrested in infection foci in curled root hairs (Fig. 1 A and B); (ii) some nodules had abnormal infections that appeared to arrest but restart from pockets of large accumulations of bacteria (Fig. 1E); and (iii) although most of the nodules were small and white (Fig. S1B), after 4–5 wk, some were larger and somewhat pink, suggesting they might be infected (Fig. S1C). The total number of ITs in the mutant was greatly reduced (Fig. 1F); a few infections were found that did progress down root hairs (Fig. 1C), but the continued growth of these infections was often abnormal within nodules (Fig. 1E). The net effect was a reduction in rhizobial infections and the formation of white nodules that were probably uninfected (Fig. 1 E and G).
Fig. 1.
Phenotype of the SL5711-2 mutant. (AC) Confocal microscopy of root hairs of WT (A) and mutant (B and C) seedlings inoculated with M. loti carrying GFP. Long ITs in the mutant, as shown in C were uncommon. (D and E) Nodules of WT (D) and mutant (E) stained with X-Gal 2 wk after inoculation with M. loti carrying lacZ. (F) Average numbers (±SE) of ITs and infection foci in WT and the mutant 1 and 2 wk after inoculation with lacZ-marked M. loti. (G) Average numbers (±SE) of mature nodules and white nodules on WT and mutant scored 3–6 wk after inoculation (±SE n > 20). (Scale bars in AC, 12 μm and in D and E, 0.2 mm.)

Identification of a Mutation in a Predicted Pectate-Lyase Gene.

The mutant (SL5711-2) was crossed with MG20, and of 2,044 F2 progeny, 486 showed the mutant phenotype, consistent with inheritance of a recessive mutation. The mutation mapped between markers TM0689 and TM1261 on the short arm of linkage group VI (Fig. S2A), but no assembled DNA sequence of this region of L. japonicus was available. TM0689 and TM1261 are located on the BAC clones LjT34N14 and LjT45M05, respectively; the DNA sequences of these BACs were searched against the Medicago truncatula genome, identifying homology with the BACs mth2-1713 and mth2-71j12, respectively. These two BACs overlap with either side of the BAC mth2-21i11, and the DNA sequence of this region of M. truncatula chromosome 3 has been determined. The markers TM0689 and TM1261were aligned with the M. truncatula sequence, identifying 38 predicted genes between the two markers (Fig. S2A). The expression patterns of these 38 genes were assessed using the M. truncatula Gene Atlas database (22) and one was expressed specifically in nodules (Fig S2B). This gene encodes a predicted pectate lyase and is strongly induced 4 d after inoculation. The predicted coding sequence (Medtr3g086320) of 1,323 bp was searched against the L. japonicus DNA database (http://www.kazusa.or.jp/lotus/index.html), identifying 86% identity over 651 bp with a short genomic fragment (LjSGA_015164). The 5′ end of the region was amplified from genomic DNA of L. japonicus Gifu and sequenced. On the basis of this sequence, the cDNA was cloned by RT-PCR and sequenced, identifying three introns (Fig. 2A) and a predicted protein of 400 residues that contains the highly conserved domains of pectate lyases (Fig. 2B and Fig. S3). Quantitative RT-PCR showed that this L. japonicus gene, like the M. truncatula gene, had higher expression in nodules than in root, leaf, stem, or flower tissue (Fig. S2C). These results, together with the syntenic location and 83% identity with the M. truncatula gene, suggested orthology.
Fig. 2.
Genetic and biochemical characterization of pectate lyase. (A) The L. japonicus pectate lyase LjNPL gene is depicted showing the exons (black boxes), the locations of npl mutations, and the locations of the primers used (short arrows) in the ChIP experiments in Fig. 4. (B) The LjNLP protein structure shows the N-terminal signal (SP) and the two regions (shaded) most highly conserved with other pectate lyases; the locations of the protein changes induced by the mutations are indicated. (C and D) A. rhizogenes induced hairy roots on Ljnpl-2 transformed with the vector control (C) or LjNPL (D). (E) SDS/PAGE of the WT and LjNPL-1 His-tagged pectate lyases purified from yeast. (F) Pectate lyase-specific activities (±SD) of the WT and LjNPL-1 proteins assayed using polygalacturonic acid and pectin (20–35% esterified) as substrates. Different letters above the bars indicate that the differences are significant (P = 0.05) on the basis of a Student's t test.
The DNA sequence of the predicted pectate lyase gene from the mutant identified a G-to-A mutation causing a G200R change in the pectate lyase (Fig. 2 A and B and Fig. S3). We named this gene LjNPL for L. japonicus nodulation pectate lyase and the allele Ljnpl-1.

Mutation of the Pectate Lyase Causes the Infection Defect.

A mutation (itd1) causing infection defects in L. japonicus was previously mapped to linkage group VI (23). From three independent crosses between Ljnpl-1 and itd1, 13 F1 seeds were obtained. All had the mutant phenotype, and so Ljnpl-1 and itd1 must be allelic. We sequenced LjNPL from itd1 and found a G-to-A mutation that changed the W343 codon to a stop (Fig. 2 A and B and Fig. S3); therefore, we redesignated the allele npl-2. We reconfirmed the npl-2 phenotype and by quantifying the number of ITs showed the phenotype to be somewhat more severe than that of npl-1; whereas with npl-1 a few ITs were observed 7 d after inoculation (Fig. 1F), none was observed with npl-1; even 2 wk after inoculation only two infections were observed on seven plants. Although most (>95%) nodules were small and white with npl-2 (Fig. S1D), after longer incubation occasional larger nodules were formed (Fig. S1E).
The wild-type pectate lyase genes were amplified from genomic DNA of both L. japonicus and M. truncatula. The L. japonicus gene (LjNPL) was cloned behind the ubiquitin promoter in pUB-GW-GFP. The M. truncatula gene (MtNPL) was cloned in pKGW-R with 2 kb of DNA upstream and 1 kb downstream of the translation start and stop. Agrobacterium rhizogenes carrying each of these constructs was used to form transformed hairy roots on chimeric plants of npl-1 and npl-2. All four transformations produced hairy roots that formed mature pink nodules 3 wk after inoculation with M. loti, very clearly showing complementation (Table 1 and Fig. 2D and Fig. S4); only 2 of 18 npl1-1 plants transformed with the vector control formed larger and somewhat pink nodules (one on each plant).
Table 1.
Complementation of Ljnpl mutants by hairy root transformation
LineTransformation constructTransgenic plants nodulated/total
Wild typeMtNPL8/9
Wild typeUbpro::LjNPL20/20
Ljnpl-1Empty vector2/18
Ljnpl-1MtNPL8/16
Ljnpl-1Ubpro::LjNPL28/44
Ljnpl-2Empty vector0/16
Ljnpl-2MtNPL6/15
Ljnpl-2Ubpro::LjNPL16/36
These data demonstrate that the pectate lyase genes LjNPL and MtNPL are orthologs and that the LjNPL is required for most root-hair infections.

Purified LjNPL Has Pectate Lyase Activity.

The predicted NPL-encoded pectate lyases from L. japonicus and M. truncatula show high similarity (81 and 83% identity, respectively) to a possible ortholog (Glyma11g37620) in soybean and 67% identity to a biochemically characterized pectate lyase (24) from Zinnia elegans (GenBank CAA70735) (Fig. S3). All of these contain characteristic regions (25) conserved in pectate lyases (Fig. 2B and Fig. S3) and a signal peptide predicted using SignalP (26) to be involved in secretion (probability = 0.990). We constructed C-terminally His-tagged derivatives using LjNPL cDNA from WT and npl1-1 and expressed them in yeast under the control of the GAL1 promoter. Both proteins were purified and eluted with imidazole from a nickel affinity column. SDS gel electrophoresis revealed proteins of about 44 kDa that were relatively free from contaminants (Fig. 2E).
Pectate lyases degrade polygalacturonic acid and pectin (methyl esterified PGA) via a β-elimination reaction that produces a product that absorbs light at 230–235 nm. This assay showed that the purified WT LjNPA degraded both PGA and pectin, showing significantly lower activity with the pectin. The mutant protein had no, or very low levels of activity with both substrates (Fig. 2F). This demonstrates that LjNPL encodes a functional pectate lyase. The absence of this pectate lyase would explain the lack of infections in the mutant (Fig. 1F), implying that the pectate lyase is required for the cell-wall degradation that occurs during the initiation of root-hair infection by rhizobia.

Analysis of Root Hair Deformation and Infections in npl-1 and npl-2 Nodules.

Pectate lyase could, in theory be associated with loosening of the cell wall to allow root hair deformation, but we were unable to distinguish any difference between Nod factor-induced root-hair deformation in the WT and the Ljnpl-2 mutant (Fig. S5). This is consistent with the presence of many infection foci in curled root hairs of both Ljnpl mutants.
Four to 5 wk after inoculation, most nodules on Ljnpl-1 and Ljnpl-2 were small, white, and uninfected, but a few were larger and slightly pink (Fig. 1F and Fig. S1 C and E). Because these nodules might be infected, we were interested to determine whether the absence of pectate lyase activity blocked the formation of intracellular infection threads within nodules. Light microscopy revealed that, compared with WT, relatively few nodule cells were infected in the mutants (Fig. 3 A and B and Fig. S6A) and that very large intercellular infection structures were present in the mutants (Fig. 3B and Fig. S6 A, C, and D). Comparison of the number of infection threads in the few infected nodules of the mutants compared with WT is difficult, because in the mutants, the nodules had to be assayed 4–5 wk after inoculation, whereas few are found in the WT at this time point. Nevertheless, in the mutants, most of these delayed infections are associated with abnormal and large infection events even though some relatively normal infection threads are present. Electron microscopy also revealed the presence of some relatively normal intracellular and intercellular infection threads in Ljnpl-2 nodules (Fig. 3 C and D). In addition, bacteroids were present in the infected cells (Fig. 3D and Fig. S6B); we found no statistically significant difference in bacteroid numbers per unit area in infected cells. These observations suggest that normal progression of infection threads in nodules is abnormal, but once entry is made into a plant cell, subsequent infection is relatively normal.
Fig. 3.
Infection events in the few large nodules formed by pectate lyase mutant. (A and B) Light micrographs of stained nodules of WT (A) and a pale pink nodule formed on Ljnpl-2 (B). The black arrows in A and B highlight infection threads and the gray arrow indicates a large abnormal infection. (C and D) Electron microscopy of nodule sections of WT (C) and Ljnpl-2 (D) showing intracellular infection threads with infection droplets following labeling with immunogold particles (black dots) using the polygalacturonic acid-specific antibody JIM5. The reduced immunolabeling around the infection droplets is marked with arrows. (Scale bars in A and B, 0.2 mm; C and D, 1 μm.)
To determine whether there were differences in pectin in infection threads in nodules, we did immunogold labeling using the pectin-specific monoclonal antibody JIM5. This identified pectin was associated with intercellular ITs in both the WT and mutant nodules (Fig. 3 C and D) and an intracellular IT in the Ljnpl-2 mutant had a normal level of JIM5-detected pectin (Fig. S6B). As illustrated with the Ljnpl-2 mutant (Fig. 3D), the density of the immunolocalization around infection droplets was decreased (arrowed) compared with the labeling of the adjacent cell wall; this decrease was similar to what was observed with the WT (Fig. 3C) implying that the pectin can be degraded at this stage. These data show that LjNPL is required for most normal infection events, but when the ITs enter individual nodule cells, normal numbers of bacteria can be released.

NPL Induction Requires NIN and Nod-Factor Activation of the Nodu-lation Signaling Pathway.

Nod-factor induction of NPL expression was observed in WT roots; mutations in the Nod-factor receptor genes (NFR1 and NFR5) and in components of the Nod-factor signaling pathway (SYMRK, CCaMK, and CYCLOPS) blocked this induction (Fig. 4A). NSP1 and NIN encode transcription factors required for nodulation and infection. The nsp-1 and nin-1 mutations reduced NPL expression, although a low level of induction was consistently observed (Fig. 4A). To test whether NIN and/or NSP1 might directly regulate NPL expression, we tested whether these proteins could bind to the NPL promoter. NSP1 had no effect on the elecrophoretic mobility of the NPL promoter, whereas the DNA-binding domain of NIN had a strong effect (Fig. 4B). This interaction was confirmed by chromatin immunoprecipitation (Fig. 4C), using antiserum to the M. truncatula NIN DNA-binding domain, which is 91% identical in sequence to that of L. japonicus NIN. The LjNPL promoter was detected in the immunoprecipitate of WT L. japonicus, but not from the nin-1 mutant. As a control for promoter specificity, we checked for coprecipitation of the 3′ end of the coding region of NPL, but saw no NIN-dependent product (Fig. 4C).
Fig. 4.
LjNPL induction requires Nod-factor signaling and NIN. (A) Induction of LjNPL by Nod factor (10 nM) relative to untreated controls was measured by quantitative RT-PCR using RNA isolated from WT and the nodulation mutants indicated. (B) Autoradiograph showed that migration of the P32-labeled LjNPL promoter in nondenaturing gel electrophoresis was not affected by added NSP1 protein, but was affected by the DNA binding domain of NIN. (C) Chromatin immunoprecipitation with NIN antiserum (anti-NIN) in WT and the Ljnin-1 mutant produced a NIN-specific interaction with the LjNPL promoter (LjNPL Pro) but not with part of exon 4 (LjNPL exon). The controls (input) before immunoprecipitation are shown. The locations of the DNA primers used are indicated in Fig. 2. (D) A. rhizogenes-induced hairy roots of L. japonicus WT and Ljnin-1 were transformed with the promoter region of LjNPL upstream of the β-glucuronidase gene. Roots were treated with (+NF) or without (−NF) 10 nM Nod factor and stained (6 h) with X-gluc. (EG) Roots of WT transformed as in D, but with seedlings grown in clay and then inoculated with M. loti. This revealed that curled root hairs stained more strongly than most other root hairs (F and G), suggesting increased expression of LjNPL in these cells. The staining in EG was done for a shorter time (3 h) than in D (6 h), to show clearly the stronger staining of some individual root hairs. (Scale bars in D and E, 2 mm; F and G, 50 μm.)
We cloned the LjNPL promoter upstream of the β-glucuronidase (GUS) reporter gene and used A. rhizogenes to produce transformed hairy roots on L. japonicus seedlings. In the WT, there appeared to be a low level of background expression of LjNPL-GUS that was increased by Nod factor (Fig. 4D) in seedlings grown on agar. The nin-1 mutant showed minimal induction (Fig. 4D), in agreement with the quantitative RT-PCR results (Fig. 4A). With M. loti inoculated onto transformed hairy roots of chimeric plants grown in clay, there was strong induction of expression in some individual root hairs with tightly curled root hair tips (Fig. 4 EG). This implies strong induction of LjNPL in root hairs associated with initiation of rhizobial infection.

Discussion

It has been evident for many years that rhizobial infection of legume root hairs and the subsequent growth of ITs across adjacent root cells are accompanied by localized degradation of the plant cell wall (2729). The degradation of cell-wall pectin would require the action of pectate lyase and/or the sequential action of pectin methyl esterase and polygalacturonase. Rhizobium etli produces a pectate lyase (HrpW), which is likely to be targeted to plant cells via a type III secretion system (30), but HrpW is not required for nodule infection, and database searches revealed that no predicted pectate lyases are in the sequenced genome of M. loti MAAF303099. Bradyrhizobium japonicum has seed-exudate (but not flavonoid) inducible genes encoding pectin methyl esterase and polygalacturonase (31), but again our database searches indicate these genes are relatively rare in rhizobia and absent from M. loti.
All plants have pectate lyase and polygalacturonase genes that are involved in cell-wall degradation associated, for example, with fruit ripening, pollen tube and cotton fiber elongation, and cell-wall changes associated with cellular differentiation and separation (24, 32). Pathogens may even subvert plant pectate lyases to enhance pathogenicity (33). In legumes, predicted pectate lyases and polygalacturonase have been described to be induced during early stages of symbiosis (19, 34). Whether these, or rhizobial cell-wall degrading enzymes are required to promote the localized cell-wall degradation required for infection has been an open question. Until recently, no bacterial or plant mutants have been identified as shedding light on this process. The work described here demonstrates that a legume pectate lyase (LjNPL) is essential for normal initiation of infection. Recent work with Rhizobium trifolii demonstrated that a nonpolar deletion of a cellulase gene blocked normal infection of clover, demonstrating a role for cellulose degradation (17, 35). However, that cellulase gene is embedded within an operon involved in bacterial cellulose synthesis and its predicted signal sequence strongly suggests that the cellulase is targeted to the periplasm. During its synthesis in bacteria, cellulose is continuously polymerized and then cleaved in the periplasm. It has been demonstrated that rhizobially made cellulose can impair the infection of root hairs (36). Therefore, it is possible that uncleaved cellulose made by the cellulase mutant could inhibit bacterial infection and so it seems an open question as to whether rhizobial cellulase is required for infection. The observation that polar mutations in the cellulose synthase operon expected to block, or greatly reduce, expression of the cellulase were not observed to affect infection (36, 37) suggests that the principle role of the rhizobial cellulase is not the degradation of plant cell wall to permit infection.
Although severe, the block of infection caused by the Ljnpl mutations was not quite complete, with some infections being produced. With the weaker allele caused by the missense mutation Ljnpl-1, this could be explained by some residual enzyme activity, but this explanation seems unlikely with the Ljnpl-2 allele, which introduces a stop codon that would stop translation of the last 57 residues of the protein. In plants there are many pectate lyases, 26 predicted in Arabidopsis thaliana (38). We identified 17 predicted pectate lyases in M. truncatula, two of which (Medtr3g086320 and Medtr3g070740) are closely homologous to LjNPL (Fig. S7); both genes are on linkage group 3 and Medtr3g086320 is the LjNPL ortholog identifed from positional cloning (Fig. S2). Glycine max also contains two LjNPL-like genes (Glyma11g37620 and Glyma18g01570) (Fig. S7), but we found no close homolog from L. japonicus. Rhizobial inoculation induces the two G. max genes (39) and Medtr3g086320 (Fig. S2B). Phylogeny (Fig. S7) shows that these five legume genes constitute a clade that is distinct from the other pectate-lyase–like genes in A. thaliana and M. truncatula (Fig. S7). The existence of this clade implies that these LjNPL-like genes have evolved a specialized role associated with legume infection by rhizobia. Most pectate lyase genes in A. thaliana are expressed in flowers, but the pectate lyases most closely related to LjNPL are also expressed in primary and lateral roots (40).
In addition to pectate lyases there are several pectin methyl esterases and polygalacturonases that could play a role in degradation of the cell-wall pectin. Therefore, the occasional observed infections could be due to some of these other proteins. We checked the M. truncatula Gene Atlas and found that predicted polygalacturonases (Medtr6g005630, Medtr2g032710, and Medtr5g034090) and a pectin methyl esterase (Medtr4g130790) are induced during nodulation. The induction of such genes may account for the low level of infection events that still occur in the mutants.
The presence of some infection threads in the mutants can explain the delayed appearance of slightly pink nodules, in which a few of the nodule cells were infected. These infected nodules tended to have large accumulations of bacteria within what appeared to be distended infection threads, but we also observed the presence of some relatively normal looking intracellular and intercellular ITs. There were also normal numbers of bacteroids in a few of the nodule cells. Therefore, if infection events do occur, even via very abnormal infection events, apparently normal infection and bacterial release can occur in a few cells. We had anticipated that there might be more pectin associated with the infection droplets in the Ljnpl mutants than the WT, but this did not seem to be the case. The decrease in antigenically reactive pectin around the infection droplets could be explained by pectin degradation via the action of other nodule-expressed pectate lyases or polygalacturonases. However, we cannot rule out the possibility that a lack of delivery of pectin at growing infection droplets might lead to an overall dilution of the detectable pectin.
The regulation of the NPL gene in roots appears to be complex. The major regulation is mediated via NIN, which we have shown binds directly to the NPL promoter. The observation that the nsp1-1 mutation caused a similar reduction in expression fits with the observation that NSP1 regulates the expression of NIN (41). However, the observation that there is also a low level of NIN and NSP1-independent expression that appears to require the Nod-factor signaling pathway implies that this pathway can induce NPL via a different transcription factor. Although Nod factors induced NPL in epidermal tissue, it was also evident that the presence of M. loti strongly induced NPL in single root hairs, particularly those showing tight curled root hair tips. This pattern of expression is remarkably similar to that observed with the expression of NIN (42), in good agreement with the observation that NIN regulates NPL.
In addition to the transcriptional regulation, it seems likely that there must be posttranslational regulation. The presence of a signal peptide is consistent with secretion of NPL. In addition the NPL protein must be very specifically targeted to those specific cell-wall locations where infection threads are initiated or where they cross cell–cell junctions.
A key question for future work will be to identify how NPL activity is localized to discrete regions of the plant cell wall. One model is that NPL could be secreted into vesicles and that these vesicles are specifically targeted to specific sites on the cell wall. The localized cell-wall degradation that can be induced by Nod factors even in the absence of infection threads (21, 29) implies a unique and specific targeting mechanism. Vesicle-associated SNARE proteins that target vesicles to specific compartments in the nodule have been identified (4345) and there is a nodule-specific protein secretory pathway that is required for bacteroid development (46). However, mechanisms for specific targeting of proteins during infection thread initiation have not been identified; the localized cell-wall degradation induced by Nod factors (21, 29) implies that this degradation can occur without rhizobia being present. NPL is probably only one of several proteins specifically associated with infection thread initiation and we believe that this work will pave the way toward identifying other localized cell-wall degradation and synthesis enzymes and an understanding of their targeting.

Materials and Methods

Plant growth, inoculation with lacZ-marked M. loti R7A, and phenotype scoring were as described previously (23); details are in SI Materials and Methods. The mapping details are in Fig. S2. Genomic DNA of LjNPL and MtNPL was cloned in pUB-GW-GFP and pKGW-R, respectively, to form red and green fluorescence-marked constructs for hairy root transformation by A. rhizogenes. For analysis of LjNPL-GUS induction by Nod factor, chimeric plants with transformed hairy roots were grown on agar medium and exposed to 10 nM Nod factor for 24 h before staining. For LjNPL-GUS induction by M. loti, chimeric plants with transformed roots were transplanted to Seramis clay watered with FP medium (18); after 3 d of growth, the plants were inoculated with M. loti and after 3 d, the roots were stained for β-glucuronidase activity. For protein purification, C-terminally His-tagged derivatives of NPL from WT and npl-1 were expressed in yeast using cDNA cloned in pYES 2 (SI Materials and Methods). Pectate lyase activity was assayed at pH 8.8 and 40 °C as described (47) using pectin (20–34% methylated) or polygalacturonic acid monitoring absorbance at 235 nm and expressing activity as nanomoles product per min per mg added protein.
To analyze LjNPL expression, seedlings were grown on agar and treated for 24 h with 1 mL of 10 nM Nod factor as described (3). For analysis of tissue-specific expression, plants were grown in vermiculite and perlite (SI Materials and Methods). RNA extraction and quantitative RT-PCR (48) were done using SYBR GREEN Master mix (Sigma) and analyzed using a CFX96 Real-Time system (Bio-Rad) over 40 cycles of 94 °C for 30 s, 63 °C for 30 s, and 72 °C for 30 s after an initial denaturation at 95 °C for 4 min. The internal controls were the genes for EF-1α as described. Data from three technical replicates and three biological repeats were analyzed using the 2-ΔΔCt method.
Assays of protein interactions with the LjNPL promoter were done using NSP1 fused to glutathione transferase and the DNA binding domain of NIN carrying a poly-His tag using proteins purified from Escherichia coli BL21 and DH5α, respectively. The plasmids, purification with glutathione agarose or Ni-NTA beads, protein quantification, and gel retardation assays were as described previously (41). Chromatin immunoprecipitation (41) was done using a ChIP kit (Millipore) with rabbit antiserum raised against the NIN peptide CRQHGITRWPSRK. Parts of the NPL promoter and fourth exon (Fig. 2A) were amplified before and after immunoprecipitation using 37 PCR cycles with the primers described in Table S1.

Data Availability

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. JN621897, JQ081955, JQ081956, and JQ081957).

Acknowledgments

We thank Tracey Welham, Trevor Wang, and Martin Parniske for making available the mutants and for help with the preliminary mutant screen; Keith Roberts for advice on pectate lyase assays; Nick Brewin and Janine Sherrier for helpful discussions; Sue Bunnewell, Kim Findlay, and Grant Calder for expert assistance with microscopy; and Akira Miyahara for advice on protein purification. The work was supported by Grant E017045/1 and a Grant-in-Aid from the Biotechnology and Biological Sciences Research Council, and by the John Innes Foundation.

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Information & Authors

Information

Published in

The cover image for PNAS Vol.109; No.2
Proceedings of the National Academy of Sciences
Vol. 109 | No. 2
January 10, 2012
PubMed: 22203959

Classifications

Data Availability

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. JN621897, JQ081955, JQ081956, and JQ081957).

Submission history

Published online: December 27, 2011
Published in issue: January 10, 2012

Keywords

  1. Medicago truncatula
  2. Mesorhizobium
  3. pectin
  4. polygalacturonase

Acknowledgments

We thank Tracey Welham, Trevor Wang, and Martin Parniske for making available the mutants and for help with the preliminary mutant screen; Keith Roberts for advice on pectate lyase assays; Nick Brewin and Janine Sherrier for helpful discussions; Sue Bunnewell, Kim Findlay, and Grant Calder for expert assistance with microscopy; and Akira Miyahara for advice on protein purification. The work was supported by Grant E017045/1 and a Grant-in-Aid from the Biotechnology and Biological Sciences Research Council, and by the John Innes Foundation.

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

Fang Xie
John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom
Jeremy D. Murray
John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom
Jiyoung Kim
John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom
Anne B. Heckmann
John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom
Anne Edwards
John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom
Giles E. D. Oldroyd
John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom
J. Allan Downie1 [email protected]
John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom

Notes

1
To whom correspondence should be addressed. E-mail: [email protected].
Author contributions: F.X. and J.A.D. designed research; F.X., J.K., A.B.H., and A.E. performed research; G.E.D.O. contributed new reagents/analytic tools; F.X., J.D.M., and J.A.D. analyzed data; and F.X. and J.A.D. wrote the paper.

Competing Interests

The authors declare no conflict of interest.

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    Legume pectate lyase required for root infection by rhizobia
    Proceedings of the National Academy of Sciences
    • Vol. 109
    • No. 2
    • pp. 347-645

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