Srsymrk, a Plant Receptor Essential for Symbiosome Formation

SrSymRK, a plant receptor essential for symbiosome formation

Ward Capoen*, Soﬁe Goormachtig*, Riet De Rycke, Katrien Schroeyers, and Marcelle Holsters†

Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium

Communicated by Marc C. E. Van Montagu, Ghent University, Ghent, Belgium, May 23, 2005 (received for review March 31, 2005) The symbiosis between legumes and rhizobia is essential for the domains bind peptidoglycan or chitin (10, 11); hence, plant nitrogen input into the life cycle on our planet. New root organs, LysM-receptor-like kinases are good candidates for NF binding the nodules, are established, which house N2-ﬁxing bacteria inter- and perception. nalized into the host cell cytoplasm as horizontally acquired or- In addition, other NF signal transduction elements act down- ganelles, the symbiosomes. The interaction is initiated by bacterial stream from the LysM-receptor-like kinases, because the cor- invasion via epidermal root hair curling and cell division in the responding mutants still respond to NFs by root hair swelling, but cortex, both triggered by bacterial nodulation factors. Of the no longer show a root hair curling (RHC) response. In Medicago several genes involved in nodule initiation that have been identi- truncatula, the Dmi1, Dmi2, and Dmi3 (12) genes encode a ﬁed, one encodes the leucine-rich repeat-type receptor kinase putative ligand-gated cation channel, a leucine-rich repeat SymRK. In SymRK mutants of Lotus japonicus or its orthologs in (LRR)-type receptor kinase, and a putative calcium and cal- Medicago sp. and Pisum sativum, nodule initiation is arrested at the modulin-dependent protein kinase, respectively (13–15). Or- level of the root hair interaction. Because of the epidermal block, thologs of Dmi2 have been described in M. sativa (MsNork), the role of SymRK at later stages of nodule development remained Lotus japonicus (LjSymRK), and Pisum sativum (Pssym19) (13, enigmatic. To analyze the role of SymRK downstream of the 16). Remarkably, mutations in these genes also impair arbuscu- epidermis, the water-tolerant legume Sesbania rostrata was used lar mycorrhization, which is an ancient and widespread endo- that has developed a nodulation strategy to circumvent root hair symbiosis that facilitates phosphate acquisition and involves 80% responses for bacterial invasion. Evidence is provided that SymRK of the land plants and fungi belonging to the Glomeromycota plays an essential role during endosymbiotic uptake in plant cells. (17). Hence, the pathways of nodulation and mycorrhization share common genetic elements for the establishment of endo- bacterial uptake ͉ endosymbiosis ͉ legume nodulation ͉ Sesbania symbiotic structures (17). Recently, a nonsymbiotic, NF- rostrata ͉ infection thread structure independent phenotype of enhanced touch sensitivity has been observed for the MsNork, the MtDmi2, and the LjSymRK mutant eguminous plants can engage into a symbiotic interaction lines. When care is taken not to disturb root hairs during Lwith rhizobia and form new root organs, the nodules, in which experimental manipulations, they curl upon application of rhi- the bacteria reside and fix atmospheric dinitrogen. Rhizobia zobia, but the curling is arrested when the root hair tip touches belong to diverse phylogenetic taxa but have in common the its own shank. Hence SymRK and its relatives are not needed for capacity to induce nodules on the appropriate legume host. The the curling process itself, although they do remain necessary for nodulation process is initiated by a complex signal exchange downstream, NF-dependent gene expression (18). We have investigated the role of SrSymRK, the unique between both partners. The principal signal molecules, the ͞ bacterial nodulation factors (NFs), are lipochitooligosaccharides Sesbania rostrata ortholog of MsNork LjSymRK in the estab- decorated with specific chemical groups at both the reducing and lishment of lateral root base (LRB) nodules. The tropical legume nonreducing end (1). Recognition of compatible NFs triggers S. rostrata is adapted to nodulate in temporarily flooded habitats developmental and cellular host responses, such as rapid ion and has versatile nodulation features. Under well-aerated con- fluxes, membrane depolarization, calcium oscillations, cytoskel- ditions, nodules form in the zone of developing root hairs via etal reorganization, root hair deformations and curling, gene RHC invasion of the microsymbiont Azorhizobium caulinodans expression, and cell division (2, 3). (19). However, on hydroponic roots, N2-fixing nodules develop Rhizobial invasion usually takes place via root hairs. Upon NF at LRBs (19). Azorhizobia invade the cortex intercellularly via perception, developing root hairs deform and eventually curl (4). cracks, resulting from lateral root protrusion, thereby circum- The bacteria are entrapped in this curl and a microcolony is venting the epidermis (20). Cortical infection pockets (IPs) are formed by NF-dependent local cell death induction and subse- formed. Local cell wall hydrolysis and invagination of the plasma quent colonization of bacteria. Presumably, IPs function as membrane lead to the formation of a tubular structure: the signaling centers, similar to rhizobial colonies within 3D root infection thread (IT) that proceeds through the root hair and hair curls (19). From IPs, ITs guide bacteria toward the target underlying cell layers to guide the bacteria toward a nodule cells for symbiotic uptake. A very comparable process takes primordium, which develops in the cortex (4). An elaborate IT place at the bases of adventitious rootlets that are located on the network spreads into the nodule primordium to provide bacteria stem of S. rostrata and that can develop into ‘‘stem’’ nodules upon to plant cells for endosymbiotic release. Uptake is initiated by inoculation. Because LRB nodulation with intercellular invasion local IT wall hydrolysis; unwalled infection droplets are formed from which bacteria are pinched off, thus creating symbiosomes

(5). Symbiosomes contain one or a few bacteria, now called Abbreviations: dpi, days postinoculation; IP, infection pocket; IT, infection thread; NF, bacteroids, surrounded by a plant-derived membrane, the perib- nodulation factor; LRB, lateral root base; LRR, leucine-rich repeat; RHC, root hair curling; acteroid or symbiosome membrane (6). LysM, lysin motif; RNAi, RNA interference. Considerable progress has been made in analyzing the plant Data deposition: The sequence reported in this paper has been deposited in the GenBank mechanisms that regulate initiation and development of nodules database (accession no. AY751547). and control of bacterial invasion. Several candidate NF receptors *W.C. and S.G. contributed equally to this work. † have been identified, which are all receptor-like kinases with To whom correspondence should be addressed. E-mail: [email protected]. PLANT BIOLOGY extracellular lysin motif (LysM) domains (7–9). Bacterial LysM © 2005 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504250102 PNAS ͉ July 19, 2005 ͉ vol. 102 ͉ no. 29 ͉ 10369–10374 Downloaded by guest on September 29, 2021 Fig. 1. SrSymRK, the SymRK ortholog of S. rostrata.(A) Sequence of the SrSymRK protein and orthologs. SrSymRK shows a 90% and 86% similarity to L. japonicus LjSymRK and M. truncatula MtDMI2, respectively. Conserved amino acids are highlighted. (B) Southern analysis with the extracellular domain of SrSymRK as a probe.

strictly depends on NFs (21), it is an excellent tool to analyze the GAAGTGA-3Ј) and SrNORKrtR2 (5Ј-CTTCGCTCATGTGT- requirement for and consequences of NF-linked perception- TGGTTGC-3Ј) primers. transduction systems in cell layers, which are deeper than the epidermis. Here, we demonstrate that RNA interference Transgenic Roots. To produce the knockout constructs (RNAi)-mediated knockdown of SrSymRK inhibits release of (pK7GWIWG27F2-SymRKKO1 and pK7GWIWG27F2-Sym- bacteria from infection droplets into the plant cytoplasm. RKKO2), two nonoverlapping regions of the LRR domain were recombined in the pK7GWIWG27F2 binary GATEWAY vec- Materials and Methods tors (Invitrogen) (27). For the pK7GWIWG27F2-SymRKKO1 Plant and Bacterial Material. S. rostrata Brem and A. caulinodans and pK7GWIWG27F2-SymRKKO2 constructs, the primers Ј ORS571(pRG960SD-32) were grown as described (22). SrSymRKRTF1attB1 (5 -GGGGACAAGTTTGTACAAA- AAAGCAGGCTCCAACCAAACAGACGTGGAAGTGA- Ј Ј Southern Analysis. DNA gel blot analysis was performed as 3 ) and SrSymRKR13attB2 (5 -GGGACCACTTTGTACA- AGAAAGCTGGGTCACTGGAAGGAATTGGTCCT-3Ј); described (23). Labeled probes were generated from the extra- Ј Ј and SrSymRKF16attB1 (5 -GGGGACAAGTTTGTAC- cellular domain with SrNORKRTF1 (5 -CCAAACAGACGT- Ј Ј Ј AAAAAAGCAGGCTTGAGCCACAACAGT-3 ) and GGAAGTGA-3 ) and SrNORKRTR2 (5 -CTTCGCTCATGT- Ј Ј SrSymRKRTR2attB2 (5 -GGGGACCACTTTGTACAAGA- GTTGGTTGC-3 ). AAGCTGGGTCTTCGCTCATGTGTTGGTTGC-3Ј) were

5 used, respectively. S. rostrata embryonic axes were transformed Identification of the Full-Length SrSymRK Sequence. Plaques (10 )of as described (28). a ␭-fix II S. rostrata genomic library were screened with a probe corresponding to the extracellular and kinase domains of M. Light Microscopy and Transmission EM. Staining with ␤-glucuroni- truncatula DMI2 (23). The full-length cDNA sequence was dase was as described (21). For semithin sections, the tissues Ј Ј obtained by 5 and 3 RACE (Smart RACE cDNA amplification were embedded in Technovit 7100 (Heraeus), cut, and stained kit, Clontech). The fragments were cloned in a pGEMTeasy with toluidine blue (20, 28). GFP analysis (28) and transmission vector (Promega). EM (21) were performed as described.

Expression Analysis. In situ hybridization was as described (24). Results The LRR region was used as template to produce 35S-labeled SrSymRK Transcripts Accumulate in the Infection Zone of LRB Nodules. antisense probe. RT-PCR was performed (25, 26) with specific The full-length cDNA of SrSymRK was obtained by genomic probes generated by SrNORKrtF1 (5Ј-CCAAACAGACGTG- library hybridization and RT-PCR (see Materials and Methods)

10370 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504250102 Capoen et al. Downloaded by guest on September 29, 2021 Fig. 2. Expresssion analysis of SrSymRK.(A) RT-PCR analysis. SrSymRK expression levels are shown for leaves, flowers, uninfected adventitious root primordia (primordium), in adventitious root primordia infected with ORS571 after 2, 4, and 6 dpi, in hydroponic roots (TϪ), and in hydroponic roots 12, 24, 36, 72, and 84 h after inoculation. (B) Bright-field image of an in situ mRNA localization in a semithin section through a nodule primordium 72 h after inoculation. (C) Dark-field image of B; signal is visible as silver grains in the infection zone. ip, infection pocket; iz, infection zone; m, meristem. (Scale bars, 100 ␮m.)

(GenBank accession no. AY751547). SrSymRK is 86% and 90% similar at the amino acid level to MtDmi2 and LjSymRK, Fig. 3. Knockdown phenotype of SrSymRK.(A) Low magniﬁcation view of a respectively (Fig. 1A). Southern hybridization with the extracel- SrSymRK knockdown line, 10 dpi. Bacteria are labeled with ␤-glucuronidase. lular domain (Fig. 1B) of MtDmi2 as probe revealed one band (B) Low magniﬁcation view of a functional WT nodule, 10 dpi. Bacteria are in each digest, demonstrating that SrSymRK is a unigene. labeled with ␤-glucuronidase. (C) Semithin section through A. IPs, ITs, and a RT-PCR was performed to analyze the expression levels of primordium are formed, but no cells are infected. (D) Semithin section SrSymRK during LRB nodulation. cDNA was prepared from through B, 10 dpi. (E and F) Details of C and D. Uninfected and infected cells stem-located adventitious root primordia before and at 2, 4, 6, are marked by triangles and asterisks, respectively. ct, central tissue; ip, and 15 d postinoculation (dpi) with A. caulinodans (Fig. 2A). infection pocket; it, infection thread; iz, infection zone; m, meristem. (Scale bars, 100 ␮m.) As seen in Fig. 2A, a basal expression was observed in adventitious root primordia, the expression was rapidly in- duced between 2 and 4 dpi, and the transcript levels dropped taining RNAi constructs developed small, white, nodule-like again in mature nodules at 15 dpi. Similarly, but less pro- structures at the base of the lateral roots of hydroponically grown nounced, SrSymRK transcripts accumulated during LRB nod- roots (Fig. 3A). Infection with ORS571(pRG960–32), which can ulation of hydroponic roots. Uninfected roots and infected be visualized by ␤-glucuronidase staining, revealed that the LRBs were harvested 12, 24, 36, 72, and 84 h after inoculation. primary invasion into the infection center was not hampered, but RT-PCR analysis showed that the transcript levels were al- spreading into the cells of the nodule primordium was inhibited. ready slightly elevated 12 h after inoculation. SrSymRK levels In contrast, the central tissue of the stained control nodules was were low in flowers and leaves (Fig. 2A). completely invaded (compare Fig. 3 A with B). RT-PCR analysis To analyze the transcript localization during nodule develop- of clonally propagated offsprings of the nine lines showed a ment, developing adventitious root nodules were hybridized in down-regulation of 65–92% in SrSymRK transcript levels when situ at 3 dpi. At this stage, ITs have reached the nodule primordium, which has the shape of an open basket with a compared to control lines (data not shown). meristem and an infection zone where bacteria are released into Microscopic analysis of semithin, toluidine blue-stained sec- plant cells. SrSymRK transcripts were detected in the infection tions through a nodule-like structure derived from a SrSymRK zone of the developing nodule (Fig. 2 B and C). RNAi line at 10 dpi revealed a nodule primordium in the typical open-basket conformation as is normally seen for WT nodules Knockdown of SrSymRK Affects Bacterial Release from ITs. To inves- at 3–6 dpi. IPs were visible, with ITs progressing toward an tigate the role for SrSymRK in LRB nodulation, two independent infection zone adjacent to the meristem (Fig. 3 C and E). RNAi constructs were made, each with a 200-bp fragment of the Nevertheless, no fixation zone with infected cells was observed SrSymRK LRR-coding sequence. These constructs were intro- but, instead, a zone of differentiated cells that were vacuolated duced into S. rostrata via Agrobacterium rhizogenes-mediated and not filled with bacteria (Fig. 3E). In contrast, the control transformation (see Materials and Methods) with a 35S-enhanced nodules had a fully differentiated central tissue containing cells GFP as cotransformation marker (28). with N2-fixing symbiosomes (Fig. 3 D and F). In total, 91 cotransformed roots were selected for analysis. Chimeric plants with transgenic roots were inoculated with A. Ultrastructural Analysis of SrSymRK RNAi Lines Reveals Multiple caulinodans, and nodulation was scored between 8 and 14 dpi. At Nodulation Defects. To better investigate eventual morphological

this stage, control lines transformed with the empty vector, all abnormalities, in vitro-propagated transgenic roots of a 70% PLANT BIOLOGY contained round, N2-fixing nodules (Fig. 3B). Nine lines con- SrSymRK knockdown line were grafted on WT shoots of S.

Capoen et al. PNAS ͉ July 19, 2005 ͉ vol. 102 ͉ no. 29 ͉ 10371 Downloaded by guest on September 29, 2021 Fig. 4. Ultrastructural analysis of the SrSymRK knockdown phenotype. (A) Transverse section through a WT intracellular IT. (B) Transverse section through an intracellular IT in an SrSymRK knockdown background, 10 dpi. Arrowhead and arrow indicate low electron-dense rim and abnormal cell wall depositions in SrSymRK knockdown lines, respectively. (C) Section through a bag-like intracellular IT consisting of numerous branches, 10 dpi. (D) Section through an intracellular IT in an SrSymRK knockdown background. Low electron-dense rim is indicated by an arrowhead. (E) Rare release of bacteria, resulting in abnormal symbiosomes, which are indicated by arrows. (F) WT bacteroids at 6 dpi. (Scale bars, 1 ␮m.)

rostrata. This particular line showed nodule primordium and IT S. rostrata has recruited such a cortical invasion track to nodulate formation, but no bacterial uptake, in accordance with the on submergence when accumulating ethylene blocks RHC in- phenotype for SrSymRK knockdown lines described above. The vasion and root hair development (22) as well as during nodu- grafts responded to bacterial inoculations in exactly the same lation at the bases of stem-located adventitious rootlets where manner as the primary transformed roots (data not shown). Ten root hairs are absent. During LRB nodulation on S. rostrata, the days after inoculation with A. caulinodans, small, nodule-like bacteria enter directly in the cortical plant tissues and infection structures were harvested for EM analysis (Materials and Meth- proceeds intercellularly in the outer cortex, without root hair ods). High magnifications of ITs in the infection zone showed involvement, yet the process depends strictly on NFs (21). that, in contrast to WT, a low electron-dense rim was present To analyze the role of SymRK downstream of the epidermis, between the IT matrix and the IT wall (Fig. 4 A, B, and D). The the S. rostrata ortholog was isolated. SrSymRK is Ϸ85–90% rim was continuous with the low electron-dense bacterial surface similar to its relatives from other legumes. Southern hybridiza- polysaccharides. ITs seemed to lose rigidity and became bag-like tions indicated that it is a unigene and expression analysis in appearance, with unusually protruding bulges (Fig. 4 C and revealed that it could indeed function downstream of the epi- D). Extensive cell wall depositions (Fig. 4B) were also obvious dermis. RT-PCR showed that the SrSymRK transcripts were that had never been seen in WT ITs (Fig. 4A). Infection droplets up-regulated during adventitious and lateral root-based nodu- were observed, but only a few symbiosome-like structures could lation to disappear again in mature nodules. The induction level be detected in the plant cell cytoplasm. They were different from during adventious root nodulation was more pronounced, re- WT symbiosomes (Fig. 4F) and had an irregular shape (Fig. 4E). flecting the ease of specific and synchronous sampling of the The bacteroids were surrounded by a large and irregular perib- stem-located adventitious root tissues. In situ hybridization acteroid space (Fig. 4E). indicated that SrSymRK transcripts were present in cells underlying the nodular meristem, i.e., in the infection zone where Discussion bacteria will be released into the plant cell cytoplasm to differ- The search for functions involved in NF perception and signal entiate into N2-fixing bacteroids. transduction has revealed a set of genes that are required for To obtain an insight into an eventual function of SrSymRK in nodulation as well as for mycorrhization, the ancient plant-fungi the infection zone, RNAi knockdown roots were constructed endosymbiosis from which nodulation has been derived (29). We with A. rhizogenes-mediated root transformation. Lines with 4- to provide more insight into the function of SymRK, a component Ͼ10-fold reduced SrSymRK transcript levels exhibited the same of the common signal transduction cascade. impaired LRB nodulation phenotype. After 2 wk, only small, LjSymRK and its orthologs MtDmi2, MsNork, and Pssym19 nonfixing nodule-like structures were detected that were barely code for LRR-type receptor kinases, and mutations in these infected with A. caulinodans. Light microscopy and EM revealed genes cause a very early arrest and almost completely block two main defects in these nodule-like structures. First, ITs were epidermal responses to rhizobia resulting in impaired RHC (13, formed, indicating that the underlying mechanism was not 16). During mycorrhization, LjSymRK mutants are arrested at hampered, but they had an irregular appearance with protruding the level of intracellular fungal entry in the exodermis͞outer bulges containing mainly IT matrix. Also the walls of the ITs cortical cells, indicating that SymRK also functions downstream were irregular with large cell wall deposits. At the ultrastructural of the epidermis, in other plant tissues (30). The question of level, the matrix of the ITs was separated from the IT wall by a whether during nodulation SymRK plays also a role in tissues layer of low electron-dense material that seemed continuous other than the epidermis is difficult to answer in legumes that use with the low electron-dense surface polysaccharide layer of the RHC invasion. However, it can be easily addressed in host plants bacteria. A flooding of the surface polysaccharides along the that offer crack entry invasion, which avoids the epidermis and edges of the ITs could imply a change in the physico-chemical the root hairs and provides direct intercellular entry in the outer nature of the IT matrix. Cross-linking of the matrix concomitant cortex at positions of LRBs. The water-tolerant tropical legume with bacterial cell division has been proposed to be the driving

10372 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504250102 Capoen et al. Downloaded by guest on September 29, 2021 force for IT progression (5); hence, changes in the matrix function. In contrast, the well established coupling of SymRK to composition might give rise to irregular IT structures. NF signal transduction in RHC nodulation (3, 9, 12, 18, 35) A second defect is observed at the level of bacterial uptake would provide an early epidermal checkpoint, which is circum- when rhizobia are released into the plant cell cytoplasm to form vented in LRB nodulation that uses direct cortical responses. symbiosomes. This process is initiated by local hydrolysis of the Besides signaling from a distance, NFs may affect adjacent IT cell wall and formation of an infection droplet, a small bulge cells in the epidermis and during the passage of the ITs in the surrounded by a plant cell membrane containing a few bacteria cortex. In the epidermal root hairs, bacterial entry requires both and IT matrix (5). From the infection droplets, individual NF perception (presumably via LysM-type receptors) and active bacteria are released in the cytoplasm (31). As shown by SymRK (3, 7–9, 13, 16). In the infection zone, where SymRK transmission EM analysis, in the SrSymRK RNAi line, infection transcripts are up-regulated, a cell-autonomous role in uptake is droplets were formed, but hardly any individual symbiosome was plausible and presumably uncoupled from local bacterial NFs. present. The few symbiosomes that were detected in the cyto- Although genes responsible for NF production are transcribed in plasm had an irregular structure, indicating a defect in bacterial ITs and NFs have been localized in the infection zone (36), a few uptake into the plant cells, possibly as a consequence of disabled cases have been described of NF-deficient strains that could vesicle trafficking. Similar features were observed in M. trunca- reach the interior of a nodule and were internalized to fix tula (32). Hence, SymRK is involved in control of IT structure nitrogen as long as NFs were added to the root medium (refs. 21 and in bacterial uptake for symbiosome formation. and 37; J. Den Herder and M. Holsters, unpublished results). Taking into consideration these observations, the known role The NF-deficient strains NGR234 of Rhizobium sp. and of SymRK in rhizobial root hair entry and mycorrhizal invasion, USDA110 of Bradyrhizobium japonicum were able to invade and as well as recent findings on the mechanisms of invasion of nodulate Vigna unguiculata and Glycine max when NFs of biotrophic pathogens, we propose that SymRK might be part of NGR234 were added at a concentration of 10Ϫ7 to 10Ϫ6 M (37). a molecular complex for intracellular residence of endosymbi- An analogous observation has been made for the LRB nodula- onts that incorporates cell wall integrity control and targeted tion on hydroponic roots of S. rostrata by coinoculating a exocytosis (33). Intracellular penetrations of biotrophic patho- bacterial mutant with defective surface polysaccharides gens and symbiotic mycorrhiza have much in common and might (ORS571-X15), but normal NFs, and a mutant without NF depend on related programs (31). During arbuscular myccorhi- production (ORS571-V44; ref. 21). The ORS571-V44 strain zal initiation in LjSymRK mutants, the fungus invades epidermal cannot enter the host because of the absence of NFs and does not cells, but is arrested after appressoria have been formed, at the ͞ provoke any nodulation-related effect (38), whereas ORS571- intracellular passage through the exodermis outer cortex (30), X15 induces nodule primordia at LRBs, but is arrested in an arrest that might be explained by a defect in a common superficially located IPs. However, ORS571-X15 can comple- pathogen͞symbiont entry system. Moreover, part of the pheno- ment ORS571-V44 for nodule invasion and functional nodules type observed in SymRK mutants during the initial steps of the are formed, which are exclusively occupied by ORS571-V44 RHC nodulation process is related to elevated touch sensitivity bacteria (21). Presumably, NFs trigger developmental gradients (18). A possible role for SymRK in connection to the recruitment from a distance, without being required in situ at the moment of of exocytosis-mediated resistance or mechanical stress-sensing SymRK-dependent internalization. mechanisms for symbiont entry could be suppression of touch- related plant responses. In summary, SymRK is required at the heart of endosymbiosis NFs, the main bacterial signals for nodule development, in leguminous plants to release bacteria into nodule cells during trigger nodulation-related effects in the epidermis, as well as a process that might testify of common origins of bacterial and changes in cortex and pericycle cells. The latter effects are fungal endophytic lifestyles and biotrophic pathogen invasion provoked from a distance because it is very unlikely that NFs strategies. migrate through symplast or apoplast while they are immobilized in the plant cell walls (34). Hence, NFs probably activate We thank Christa Verplancke and Annick De Keyser for excellent technical assistance and Martine De Cock for careful preparation of the secondary signals that, together with signals coming from the manuscript. This work was supported by the Interuniversity Poles of stele, determine the landscape for nodule development and Attraction Program-Belgian Science Policy (P5͞13) and the Fund for preparation for invasion (2). During LRB nodulation in S. Scientific Research-Flanders (‘‘Krediet aan Navorsers’’ 1.5.088.99N and rostrata, the distant NF-dependent response of nodule primor- 1.5.192.01N). W.C. and K.S. are indebted to the Institute for the dium formation is not disturbed in roots with Ͼ10-fold SrSymRK Promotion of Innovation by Science and Technology in Flanders for silencing, suggesting that it may not be linked to SymRK predoctoral fellowships.

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