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Legume Pectate Lyase Required for Root Infection by Rhizobia

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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 accumu- lations 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 degra- dation 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.
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