Legume Pectate Lyase Required for Root Infection by Rhizobia

Legume pectate lyase required for root infection by rhizobia

Fang Xie, Jeremy D. Murray, Jiyoung Kim, Anne B. Heckmann, Anne Edwards, Giles E. D. Oldroyd, and J. Allan Downie1

John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom

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)

To allow rhizobial infection of legume roots, plant cell walls must degrade the root-hair cell wall (16, 17). Alternatively, plant cell- be locally degraded for plant-made infection threads (ITs) to be wall degrading enzymes induced in response to rhizobia may be formed. Here we identify a Lotus japonicus nodulation pectate responsible (18–20) and indeed Nod factors can promote local- lyase gene (LjNPL), which is induced in roots and root hairs by ized cell-wall degradation (21). However, there is no unequivocal rhizobial nodulation (Nod) factors via activation of the nodulation evidence as to which of these two models is correct. In this work signaling pathway and the NIN transcription factor. Two Ljnpl we demonstrate that rhizobia induce a Lotus japonicus pectate mutants produced uninfected nodules and most infections arrested lyase, which is required for root-hair and nodule infection as infection foci in root hairs or roots. The few partially infected by rhizobia. nodules that did form contained large abnormal infections. The Results purified LjNPL protein had pectate lyase activity, demonstrating fi L. japonicus that this activity is required for rhizobia to penetrate the cell wall Identi cation of an Infection Mutant of . A L. japonicus mutant (SL5711-2) defective for infection by Mesorhizobium loti and initiate formation of plant-made infection threads. Therefore, fi we conclude that legume-determined degradation of plant cell was identi ed and was of interest because: (i) unlike WT, most infections were arrested in infection foci in curled root hairs walls is required for root infection during initiation of the symbiotic (Fig. 1 A and B); (ii) some nodules had abnormal infections that interaction between rhizobia and legumes. appeared to arrest but restart from pockets of large accumulations of bacteria (Fig. 1E); and (iii) although most of the nod- Medicago truncatula | Mesorhizobium | pectin | polygalacturonase ules were small and white (Fig. S1B), after 4–5 wk, some were PLANT BIOLOGY larger and somewhat pink, suggesting they might be infected (Fig. he infection of legumes by nitrogen-fixing rhizobia occurs via S1C). The total number of ITs in the mutant was greatly reduced Tplant-made infection threads (ITs). These tube-like struc- (Fig. 1F); a few infections were found that did progress down root tures, lined with a plant cell wall and membrane, are usually ini- hairs (Fig. 1C), but the continued growth of these infections was tiated in curled root hairs and grow down through the root hair often abnormal within nodules (Fig. 1E). The net effect was and continue growing through epidermal and cortical cells (1). a reduction in rhizobial infections and the formation of white When the growing IT reaches the dividing root cells that make up nodules that were probably uninfected (Fig. 1 E and G). the nodule primordium, the plant cell wall of the IT is lost and the bacteria are budded off into the plant cytoplasm surrounded by Identification of a Mutation in a Predicted Pectate-Lyase Gene. The a plant-derived membrane. The bacteria then differentiate into mutant (SL5711-2) was crossed with MG20, and of 2,044 F2 nitrogen-fixing forms called bacteroids and in the mature nodule, progeny, 486 showed the mutant phenotype, consistent with in- they fixN2, producing ammonia that is translocated to the plant. heritance of a recessive mutation. The mutation mapped be- The initiation and growth of ITs require signaling between tween markers TM0689 and TM1261 on the short arm of linkage rhizobia and legumes. Rhizobial nodulation (Nod) factors acti- group VI (Fig. S2A), but no assembled DNA sequence of this vate nuclear-associated calcium spiking via a signaling cascade region of L. japonicus was available. TM0689 and TM1261 are that requires LysM-receptor kinases and a leucine-rich repeat located on the BAC clones LjT34N14 and LjT45M05, re- receptor-like kinase in the plasma membrane and nucleoporins spectively; the DNA sequences of these BACs were searched and ion channels in the nuclear membrane. The subsequent ac- against the Medicago truncatula genome, identifying homology tivation of a calcium and calmodulin-dependent kinase then with the BACs mth2-1713 and mth2-71j12, respectively. These activates transcription factors required for the induction of nod- two BACs overlap with either side of the BAC mth2-21i11, and ulation and infection genes (2). Nod factors also induce a calcium the DNA sequence of this region of M. truncatula chromosome 3 influx that is associated with depolarization of the plasma mem- has been determined. The markers TM0689 and TM1261were brane; this calcium influx has been proposed to be important for aligned with the M. truncatula sequence, identifying 38 predicted initiation of infection (3). Oligosaccharides derived from the genes between the two markers (Fig. S2A). The expression pat- synthesis of the rhizobial exopolysaccharide also play a crucial terns of these 38 genes were assessed using the M. truncatula role in initiation of infection, possibly by suppressing plant de- Gene Atlas database (22) and one was expressed specifically in fense responses (4, 5). Membrane-associated remorins and flo- nodules (Fig S2B). This gene encodes a predicted pectate lyase tillins that promote protein interactions and alter membrane and is strongly induced 4 d after inoculation. The predicted dynamics are also important for infection (6, 7). coding sequence (Medtr3g086320) of 1,323 bp was searched 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 Author contributions: F.X. and J.A.D. designed research; F.X., J.K., A.B.H., and A.E. per- remodeling the cytoskeleton are required for infection initiation formed research; G.E.D.O. contributed new reagents/analytic tools; F.X., J.D.M., and J.A.D. (8, 9). However, although other genes with both identified (10– analyzed data; and F.X. and J.A.D. wrote the paper. 14) and undefined roles (1) have been characterized, these genes The authors declare no conflict of interest. have not yet given insights into the mechanistic changes required This article is a PNAS Direct Submission. for initiation of root-hair infection. Data deposition: The sequences reported in this paper have been deposited in the Gen- It has been recognized for over 120 y that local penetration of Bank database (accession nos. JN621897, JQ081955, JQ081956, and JQ081957). the plant cell wall is required for legume infection (15) and there 1To whom correspondence should be addressed. E-mail: [email protected]. are two schools of thought as to how this penetration is achieved. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Bacterially produced enzymes have been proposed to locally 1073/pnas.1113992109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1113992109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 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 Fig. 1. Phenotype of the SL5711-2 mutant. (A–C) Confocal microscopy of structure shows the N-terminal signal (SP) and the two regions (shaded) most root hairs of WT (A) and mutant (B and C) seedlings inoculated with M. loti highly conserved with other pectate lyases; the locations of the protein carrying GFP. Long ITs in the mutant, as shown in C were uncommon. (D and changes induced by the mutations are indicated. (C and D) A. rhizogenes E) Nodules of WT (D) and mutant (E) stained with X-Gal 2 wk after in- induced hairy roots on Ljnpl-2 transformed with the vector control (C)or oculation with M. loti carrying lacZ.(F) Average numbers (±SE) of ITs and LjNPL (D). (E) SDS/PAGE of the WT and LjNPL-1 His-tagged pectate lyases infection foci in WT and the mutant 1 and 2 wk after inoculation with lacZ- purified from yeast. (F) Pectate lyase-specific activities (±SD) of the WT and marked M. loti.(G) Average numbers (±SE) of mature nodules and white LjNPL-1 proteins assayed using polygalacturonic acid and pectin (20–35% nodules on WT and mutant scored 3–6 wk after inoculation (±SE n > 20). esterified) as substrates. Different letters above the bars indicate that the (Scale bars in A–C,12μm and in D and E, 0.2 mm.) differences are significant (P = 0.05) on the basis of a Student’s t test.

against the L. japonicus DNA database (http://www.kazusa.or.jp/ The wild-type pectate lyase genes were amplified from geno- lotus/index.html), identifying 86% identity over 651 bp with a mic DNA of both L. japonicus and M. truncatula. The L. japo- short genomic fragment (LjSGA_015164). The 5′ end of the nicus gene (LjNPL) was cloned behind the ubiquitin promoter in region was amplified from genomic DNA of L. japonicus Gifu pUB-GW-GFP. The M. truncatula gene (MtNPL) was cloned in and sequenced. On the basis of this sequence, the cDNA was pKGW-R with 2 kb of DNA upstream and 1 kb downstream of cloned by RT-PCR and sequenced, identifying three introns the translation start and stop. Agrobacterium rhizogenes carrying (Fig. 2A) and a predicted protein of 400 residues that contains each of these constructs was used to form transformed hairy the highly conserved domains of pectate lyases (Fig. 2B and Fig. roots on chimeric plants of npl-1 and npl-2. All four trans- S3). Quantitative RT-PCR showed that this L. japonicus gene, formations produced hairy roots that formed mature pink nod- like the M. truncatula gene, had higher expression in nodules ules 3 wk after inoculation with M. loti, very clearly showing than in root, leaf, stem, or flower tissue (Fig. S2C). These results, complementation (Table 1 and Fig. 2D and Fig. S4); only 2 of 18 together with the syntenic location and 83% identity with the npl1-1 plants transformed with the vector control formed larger M. truncatula gene, suggested orthology. and somewhat pink nodules (one on each plant). The DNA sequence of the predicted pectate lyase gene from These data demonstrate that the pectate lyase genes LjNPL the mutant identified a G-to-A mutation causing a G200R change and MtNPL are orthologs and that the LjNPL is required for in the pectate lyase (Fig. 2 A and B and Fig. S3). We named this most root-hair infections. gene LjNPL for L. japonicus nodulation pectate lyase and the allele Ljnpl-1. Purified LjNPL Has Pectate Lyase Activity. The predicted NPL- encoded pectate lyases from L. japonicus and M. truncatula show 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 Table 1. Complementation of Ljnpl mutants by hairy root crosses between Ljnpl-1 and itd1,13F1 seeds were obtained. All transformation 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 Transformation Transgenic plants changed the W343 codon to a stop (Fig. 2 A and B and Fig. S3); Line construct nodulated/total fi therefore, we redesignated the allele npl-2. We recon rmed the Wild type MtNPL 8/9 npl-2 phenotype and by quantifying the number of ITs showed the Wild type Ubpro::LjNPL 20/20 phenotype to be somewhat more severe than that of npl-1; Ljnpl-1 Empty vector 2/18 whereas with npl-1 a few ITs were observed 7 d after inoculation Ljnpl-1 MtNPL 8/16 (Fig. 1F), none was observed with npl-1; even 2 wk after inocu- Ljnpl-1 Ubpro::LjNPL 28/44 lation only two infections were observed on seven plants. Al- > Ljnpl-2 Empty vector 0/16 though most ( 95%) nodules were small and white with npl-2 Ljnpl-2 MtNPL 6/15 (Fig. S1D), after longer incubation occasional larger nodules were Ljnpl-2 Ubpro::LjNPL 16/36 formed (Fig. S1E).

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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,1μm.)

high similarity (81 and 83% identity, respectively) to a possible pectin, showing significantly lower activity with the pectin. The ortholog (Glyma11g37620) in soybean and 67% identity to a mutant protein had no, or very low levels of activity with both biochemically characterized pectate lyase (24) from Zinnia ele- substrates (Fig. 2F). This demonstrates that LjNPL encodes a gans (GenBank CAA70735) (Fig. S3). All of these contain functional pectate lyase. The absence of this pectate lyase would characteristic regions (25) conserved in pectate lyases (Fig. 2B explain the lack of infections in the mutant (Fig. 1F), implying that and Fig. S3) and a signal peptide predicted using SignalP (26) to the pectate lyase is required for the cell-wall degradation that be involved in secretion (probability = 0.990). We constructed occurs during the initiation of root-hair infection by rhizobia. C-terminally His-tagged derivatives using LjNPL cDNA from WT and npl1-1 and expressed them in yeast under the control of Analysis of Root Hair Deformation and Infections in npl-1 and npl-2 the GAL1 promoter. Both proteins were purified and eluted with Nodules. Pectate lyase could, in theory be associated with loos- imidazole from a nickel affinity column. SDS gel electrophoresis ening of the cell wall to allow root hair deformation, but we were revealed proteins of about 44 kDa that were relatively free from unable to distinguish any difference between Nod factor-induced contaminants (Fig. 2E). root-hair deformation in the WT and the Ljnpl-2 mutant (Fig. Pectate lyases degrade polygalacturonic acid and pectin S5). This is consistent with the presence of many infection foci in (methyl esterified PGA) via a β-elimination reaction that pro- curled root hairs of both Ljnpl mutants. duces a product that absorbs light at 230–235 nm. This assay Four to 5 wk after inoculation, most nodules on Ljnpl-1 and showed that the purified WT LjNPA degraded both PGA and Ljnpl-2 were small, white, and uninfected, but a few were larger

Xie et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 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 obser- vations 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. 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,andCYCLOPS) 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 Fig. 4. LjNPL induction requires Nod-factor signaling and NIN.(A) Induction effect on the elecrophoretic mobility of the NPL promoter, whereas the DNA-binding domain of NIN had a strong effect (Fig. of LjNPL by Nod factor (10 nM) relative to untreated controls was measured fi by quantitative RT-PCR using RNA isolated from WT and the nodulation 4B). This interaction was con rmed by chromatin immunopre- mutants indicated. (B) Autoradiograph showed that migration of the P32- cipitation (Fig. 4C), using antiserum to the M. truncatula NIN labeled LjNPL promoter in nondenaturing gel electrophoresis was not af- DNA-binding domain, which is 91% identical in sequence to that fected by added NSP1 protein, but was affected by the DNA binding domain of L. japonicus NIN. The LjNPL promoter was detected in the of NIN. (C) Chromatin immunoprecipitation with NIN antiserum (anti-NIN) immunoprecipitate of WT L. japonicus, but not from the nin-1 in WT and the Ljnin-1 mutant produced a NIN-specific interaction with mutant. As a control for promoter specificity, we checked for the LjNPL promoter (LjNPL Pro) but not with part of exon 4 (LjNPL exon). The coprecipitation of the 3′ end of the coding region of NPL, but saw controls (input) before immunoprecipitation are shown. The locations of the no NIN-dependent product (Fig. 4C). DNA primers used are indicated in Fig. 2. (D) A. rhizogenes-induced hairy We cloned the LjNPL promoter upstream of the β-glucuron- roots of L. japonicus WT and Ljnin-1 were transformed with the promoter idase (GUS) reporter gene and used A. rhizogenes to produce region of LjNPL upstream of the β-glucuronidase gene. Roots were treated transformed hairy roots on L. japonicus seedlings. In the WT, with (+NF) or without (−NF) 10 nM Nod factor and stained (6 h) with X-gluc. – there appeared to be a low level of background expression of (E G) Roots of WT transformed as in D, but with seedlings grown in clay and LjNPL-GUS that was increased by Nod factor (Fig. 4D)in then inoculated with M. loti. This revealed that curled root hairs stained seedlings grown on agar. The nin-1 mutant showed minimal in- more strongly than most other root hairs (F and G), suggesting increased expression of LjNPL in these cells. The staining in E–G was done for a shorter duction (Fig. 4D), in agreement with the quantitative RT-PCR time (3 h) than in D (6 h), to show clearly the stronger staining of some results (Fig. 4A). With M. loti inoculated onto transformed hairy individual root hairs. (Scale bars in D and E, 2 mm; F and G,50μm.) 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 E–G). This implies strong induction of LjNPL in and slightly pink (Fig. 1F and Fig. S1 C and E). Because these root hairs associated with initiation of rhizobial infection. nodules might be infected, we were interested to determine Discussion whether the absence of pectate lyase activity blocked the formation of intracellular infection threads within nodules. Light mi- It has been evident for many years that rhizobial infection of croscopy revealed that, compared with WT, relatively few nodule legume root hairs and the subsequent growth of ITs across adjacent root cells are accompanied by localized degradation of the cells were infected in the mutants (Fig. 3 A and B and Fig. S6A) plant cell wall (27–29). The degradation of cell-wall pectin would and that very large intercellular infection structures were present require the action of pectate lyase and/or the sequential action in the mutants (Fig. 3B and Fig. S6 A, C, and D). Comparison of of pectin methyl esterase and polygalacturonase. Rhizobium etli the number of infection threads in the few infected nodules of the produces a pectate lyase (HrpW), which is likely to be targeted to fi mutants compared with WT is dif cult, because in the mutants, plant cells via a type III secretion system (30), but HrpW is not the nodules had to be assayed 4–5 wk after inoculation, whereas required for nodule infection, and database searches revealed few are found in the WT at this time point. Nevertheless, in the that no predicted pectate lyases are in the sequenced genome of mutants, most of these delayed infections are associated with M. loti MAAF303099. Bradyrhizobium japonicum has seed-exu- abnormal and large infection events even though some relatively date (but not flavonoid) inducible genes encoding pectin methyl

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1113992109 Xie et al. Downloaded by guest on September 28, 2021 esterase and polygalacturonase (31), but again our database a few of the nodule cells were infected. These infected nodules searches indicate these genes are relatively rare in rhizobia and tended to have large accumulations of bacteria within what absent from M. loti. appeared to be distended infection threads, but we also observed All plants have pectate lyase and polygalacturonase genes that the presence of some relatively normal looking intracellular and are involved in cell-wall degradation associated, for example, intercellular ITs. There were also normal numbers of bacteroids with fruit ripening, pollen tube and cotton fiber elongation, and in a few of the nodule cells. Therefore, if infection events do cell-wall changes associated with cellular differentiation and occur, even via very abnormal infection events, apparently normal separation (24, 32). Pathogens may even subvert plant pectate infection and bacterial release can occur in a few cells. We had lyases to enhance pathogenicity (33). In legumes, predicted anticipated that there might be more pectin associated with the pectate lyases and polygalacturonase have been described to be infection droplets in the Ljnpl mutants than the WT, but this did induced during early stages of symbiosis (19, 34). Whether these, not seem to be the case. The decrease in antigenically reactive or rhizobial cell-wall degrading enzymes are required to promote pectin around the infection droplets could be explained by pectin the localized cell-wall degradation required for infection has degradation via the action of other nodule-expressed pectate been an open question. Until recently, no bacterial or plant lyases or polygalacturonases. However, we cannot rule out the mutants have been identified as shedding light on this process. possibility that a lack of delivery of pectin at growing infection The work described here demonstrates that a legume pectate droplets might lead to an overall dilution of the detectable pectin. lyase (LjNPL) is essential for normal initiation of infection. The regulation of the NPL gene in roots appears to be complex. Recent work with Rhizobium trifolii demonstrated that a non- The major regulation is mediated via NIN, which we have shown polar deletion of a cellulase gene blocked normal infection of binds directly to the NPL promoter. The observation that the nsp1-1 clover, demonstrating a role for cellulose degradation (17, 35). mutation caused a similar reduction in expression fits with the ob- However, that cellulase gene is embedded within an operon in- servation that NSP1 regulates the expression of NIN (41). However, volved in bacterial cellulose synthesis and its predicted signal the observation that there is also a low level of NIN and NSP1-in- sequence strongly suggests that the cellulase is targeted to the dependent expression that appears to require the Nod-factor sig- periplasm. During its synthesis in bacteria, cellulose is continu- naling pathway implies that this pathway can induce NPL via a ously polymerized and then cleaved in the periplasm. It has been different transcription factor. Although Nod factors induced NPL demonstrated that rhizobially made cellulose can impair the in- in epidermal tissue, it was also evident that the presence of M. loti fection of root hairs (36). Therefore, it is possible that uncleaved strongly induced NPL in single root hairs, particularly those cellulose made by the cellulase mutant could inhibit bacterial showing tight curled root hair tips. This pattern of expression is remarkably similar to that observed with the expression of NIN (42),

infection and so it seems an open question as to whether rhi- PLANT BIOLOGY zobial cellulase is required for infection. The observation that in good agreement with the observation that NIN regulates NPL. polar mutations in the cellulose synthase operon expected to In addition to the transcriptional regulation, it seems likely block, or greatly reduce, expression of the cellulase were not that there must be posttranslational regulation. The presence of observed to affect infection (36, 37) suggests that the principle a signal peptide is consistent with secretion of NPL. In addition role of the rhizobial cellulase is not the degradation of plant cell the NPL protein must be very specifically targeted to those wall to permit infection. specific cell-wall locations where infection threads are initiated Although severe, the block of infection caused by the Ljnpl or where they cross cell–cell junctions. mutations was not quite complete, with some infections being A key question for future work will be to identify how NPL produced. With the weaker allele caused by the missense mu- activity is localized to discrete regions of the plant cell wall. One tation Ljnpl-1, this could be explained by some residual enzyme model is that NPL could be secreted into vesicles and that these activity, but this explanation seems unlikely with the Ljnpl-2 al- vesicles are specifically targeted to specific sites on the cell wall. lele, which introduces a stop codon that would stop translation The localized cell-wall degradation that can be induced by Nod of the last 57 residues of the protein. In plants there are many factors even in the absence of infection threads (21, 29) implies fi pectate lyases, 26 predicted in Arabidopsis thaliana (38). We a unique and speci c targeting mechanism. Vesicle-associated fi identified 17 predicted pectate lyases in M. truncatula, two of SNARE proteins that target vesicles to speci c compartments in fi – which (Medtr3g086320 and Medtr3g070740) are closely homol- the nodule have been identi ed (43 45) and there is a nodule- fi ogous to LjNPL (Fig. S7); both genes are on linkage group 3 and speci c protein secretory pathway that is required for bacteroid fi Medtr3g086320 is the LjNPL ortholog identifed from positional development (46). However, mechanisms for speci c targeting of cloning (Fig. S2). Glycine max also contains two LjNPL-like proteins during infection thread initiation have not been iden- fi genes (Glyma11g37620 and Glyma18g01570) (Fig. S7), but we ti ed; the localized cell-wall degradation induced by Nod factors found no close homolog from L. japonicus. Rhizobial inoculation (21, 29) implies that this degradation can occur without rhizobia G. max being present. NPL is probably only one of several proteins induces the two genes (39) and Medtr3g086320 (Fig. fi S2B). Phylogeny (Fig. S7) shows that these five legume genes speci cally associated with infection thread initiation and we constitute a clade that is distinct from the other pectate-lyase– believe that this work will pave the way toward identifying other like genes in A. thaliana and M. truncatula (Fig. S7). The exis- localized cell-wall degradation and synthesis enzymes and an tence of this clade implies that these LjNPL-like genes have understanding of their targeting. evolved a specialized role associated with legume infection by rhizobia. Most pectate lyase genes in A. thaliana are expressed in Materials and Methods flowers, but the pectate lyases most closely related to LjNPL are Plant growth, inoculation with lacZ-marked M. loti R7A, and phenotype also expressed in primary and lateral roots (40). scoring were as described previously (23); details are in SI Materials and In addition to pectate lyases there are several pectin methyl 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 esterases and polygalacturonases that could play a role in deg- fl radation of the cell-wall pectin. Therefore, the occasional ob- and green uorescence-marked constructs for hairy root transformation by served infections could be due to some of these other proteins. A. rhizogenes. For analysis of LjNPL-GUS induction by Nod factor, chimeric plants with transformed hairy roots were grown on agar medium and ex- We checked the M. truncatula Gene Atlas and found that pre- posed to 10 nM Nod factor for 24 h before staining. For LjNPL-GUS induction dicted polygalacturonases (Medtr6g005630, Medtr2g032710, and by M. loti, chimeric plants with transformed roots were transplanted to Medtr5g034090) and a pectin methyl esterase (Medtr4g130790) Seramis clay watered with FP medium (18); after 3 d of growth, the plants are induced during nodulation. The induction of such genes may were inoculated with M. loti and after 3 d, the roots were stained for account for the low level of infection events that still occur in β-glucuronidase activity. For protein purification, C-terminally His-tagged the mutants. derivatives of NPL from WT and npl-1 were expressed in yeast using cDNA The presence of some infection threads in the mutants can cloned in pYES 2 (SI Materials and Methods). Pectate lyase activity was explain the delayed appearance of slightly pink nodules, in which assayed at pH 8.8 and 40 °C as described (47) using pectin (20–34%

Xie et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 methylated) or polygalacturonic acid monitoring absorbance at 235 nm and Ni-NTA beads, protein quantification, and gel retardation assays were as expressing activity as nanomoles product per min per mg added protein. described previously (41). Chromatin immunoprecipitation (41) was done To analyze LjNPL expression, seedlings were grown on agar and treated using a ChIP kit (Millipore) with rabbit antiserum raised against the NIN for 24 h with 1 mL of 10 nM Nod factor as described (3). For analysis of tissue- peptide CRQHGITRWPSRK. Parts of the NPL promoter and fourth exon (Fig. fi speci c expression, plants were grown in vermiculite and perlite (SI Mate- 2A) were amplified before and after immunoprecipitation using 37 PCR rials and Methods). RNA extraction and quantitative RT-PCR (48) were done cycles with the primers described in Table S1. 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 ACKNOWLEDGMENTS. We thank Tracey Welham, Trevor Wang, and Martin for 30 s after an initial denaturation at 95 °C for 4 min. The internal controls Parniske for making available the mutants and for help with the preliminary α were the genes for EF-1 as described. Data from three technical replicates mutant screen; Keith Roberts for advice on pectate lyase assays; Nick Brewin -ΔΔCt and three biological repeats were analyzed using the 2 method. and Janine Sherrier for helpful discussions; Sue Bunnewell, Kim Findlay, and Assays of protein interactions with the LjNPL promoter were done using Grant Calder for expert assistance with microscopy; and Akira Miyahara for NSP1 fused to glutathione transferase and the DNA binding domain of NIN advice on protein purification. The work was supported by Grant E017045/1 carrying a poly-His tag using proteins purified from Escherichia coli BL21 and and a Grant-in-Aid from the Biotechnology and Biological Sciences Research DH5α, respectively. The plasmids, purification with glutathione agarose or Council, and by the John Innes Foundation.

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