The Plant Journal (2000) 22(1), 71±78
SHORT COMMUNICATION
A lipochito-oligosaccharide, Nod factor, induces transient calcium in¯ux in soybean suspension-cultured cells Tadashi Yokoyama1,*,², Naoki Kobayashi2,³, Hiroshi Kouchi1, Kiwamu Minamisawa2,§, Hisatoshi Kaku1 and Kenichi Tsuchiya1 1 National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan, and 2 Ibaraki University, Amimachi 3998, Inashiki, Ibaraki, 300-0300, Japan Received 8 September 1999; revised 8 February 2000; accepted 9 February 2000. *For correspondence (fax +81 42 367 5677; email
[email protected]). ² Present address: Tokyo University of Agriculture and Technology, Saiwaicho 3-5-8, Fuchu, Tokyo, 183-8509, Japan. ³ Present address: Miyarisan Co., Tokura 2352, Tokuramachi, Hanishinagunn, Nagano, 389-0800, Japan. § Present address: Tohoku University, Katahira 2-1-2, Aobaku, Sendai, Miyagi, 980-0812, Japan.
Summary Lipochito-oligosaccharides (Nod factors) produced by Rhizobium or Bradyrhizobium are the key signal molecules for eliciting nodulation in their corresponding host legumes. To elucidate the signal transduction events mediated by Nod factors, we investigated the effects of Nod factors on the cytosolic [Ca2+] of protoplasts prepared from roots and suspension-cultured cells of soybean (Glycine max and G. soja) using a ¯uorescent Ca2+ indicator, Fura-PE3. NodBj-V (C18:1, MeFuc), which is a major component of Nod factors produced by Bradyrhizobium japonicum, induces transient elevation of cytosolic [Ca2+] in the cells of soybean within a few minutes. This effect is speci®c to soybean cells and was not observed in the tobacco BY-2 cells. Furthermore, NodBj-V without MeFuc did not induce any cytosolic [Ca2+] elevation in soybean cells. Exclusion of Ca2+ from the medium, as well as pre-treatment of the cells with an external Ca2+ chelator or with a plasma membrane voltage-dependent Ca2+ channel inhibitor, suppressed the Nod factor-dependent cytosolic [Ca2+] elevation. These results indicate that transient Ca2+ in¯ux from extracellular ¯uid is one of the earliest responses of soybean cells to NodBj-V (C18:1, MeFuc) in a host-speci®c manner.
Introduction Soil bacteria of the genus Rhizobium, Bradyrhizobium and Azorhizobium induce the formation of root nodules on legume plants, in which the bacteria are capable of ®xing atmospheric nitrogen. The initial steps of the interactions between microsymbionts and host legumes involve the exchange of speci®c signal molecules. Lipochito-oligosaccharides (Nod factors), which are produced by bacteria in response to ¯avonoids secreted by host legumes, are the key signal molecules for eliciting nodule formation (for reviews, see Long, 1996; Spaink and Lugtenberg, 1994). Nano- to picomolar concentrations of Nod factors induce various responses in legume roots, such as deformation of root hairs (Lerouge et al., 1990), expression of early nodulin genes (Horvath et al., 1993; Minami et al., 1996; Vijn et al., 1993), formation of cytoplasmic bridges (Van Brussel et al., 1992), and cortical cell ã 2000 Blackwell Science Ltd
division leading to the formation of nodule primordia (Stokkermans and Peters, 1994; Truchet et al., 1991). However, the molecular mechanisms of Nod factor perception and Nod factor-induced signal transduction pathways in legume plants are still unknown. The earliest response of legume plants to Nod factors identi®ed so far is depolarization of transmembrane potential of alfalfa root hairs, which is observed within a minute after applying the R. meliloti Nod factor (Ehrhardt et al., 1992; Felle et al., 1995; Felle et al., 1996; Kurkdjian, 1995). Ehrhardt et al. (1996) reported a periodic oscillation in cytosolic calcium in alfalfa root hairs evoked by the R. meliloti Nod factor. This oscillation was sensitive to external calcium concentration. Gehring et al. (1997) reported that application of NodNGR(Ac,S) at 10±9 M to the homologous legume Vigna unguiculata resulted within 71
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seconds in a plateau-like increase in intercellular free calcium in root hairs. Recently, Felle et al. (1998) found a transient increase in [Cl±] and [K+], but a decrease in [H+] and [Ca2+] in the root hair zone when M. sativa was exposed to NodRm-IV (C16:2, S). Their observations suggest that the signalling cascade triggered by Nod factors involves a change in calcium ¯ux across the plasma membrane. Molecular and biochemical analyses of the Nod signal transduction pathway necessitate substantial amounts of responsive material. The host legume roots, however, contain only a few reactive cells in a limited region (in the emerging root hair zone) that is masked by the mass of non-responsive cells. Savoure et al. (1994) explored the potential use of alfalfa cell suspensions to study the expression of cell-cycle marker genes and the iso¯avonoid pathway marker gene in Medicago cells in response to NodRm-IV (C16:2, S), and reported that not only differentiated root hair cells or root cortical cells are able to respond to NodRm-IV (C16:2, S) but also Medicago cells grown in microcallus suspension (Savoure et al., 1994; Savoure et al., 1997). In this paper, we show that protoplasts generated from soybean roots or suspension cultures are responsive to Nod factors. We observed that Nod factors induce transient elevation of the cytosolic [Ca2+] in protoplasts of root cells and suspension-cultured microcallus cells of soybean plants. This response is speci®c for NodBj-V (C18:1, MeFuc), which has been demonstrated to induce root hair deformation and nodule initiation activity on soybean roots (Stokkerman and Peters, 1994), but is not observed for NodBj-V lacking the methylfucose residue. Results and Discussion To elucidate the signal transduction mediated by Nod factors, we investigated the effects of Nod factors (from B. japonicum, USDA 110) on the cytosolic [Ca2+] in protoplasts prepared from roots and suspension-cultured cells of soybean plants (Glycine max and G. soja), by means of a ¯uorescent Ca2+ indicator, Fura-PE3. Fura-2 is widely used to assay [Ca2+] in many cells. However, Fura-2 is frequently extruded from the cell (Cobbold and Rink, 1987; Tsien, 1989). Vorndran et al. (1995) reported that the availability of the zwitterionic analogue Fura-PE3 may overcome this extrusion problem. Milner et al. (1998) also studied the extrusion of Fura-2 and Fura-PE3 from platelets, and found that Fura-PE3 was extruded at a signi®cantly slower rate than Fura-2. Changes in the cytosolic [Ca2+] of protoplasts isolated from G. max root cells, G. max suspension-cultured cells and G. soja (wild soybean) suspension-cultured cells after addition of NodBj-V (C18 : 1, MeFuc) at 10±9 M (®nal concentration) are shown in Figure 1(a±c). The calibrated
base level for the calcium signal varied from experiment to experiment, as shown. We postulated that this variation resulted from a variation in cell batches. To evaluate whether the non-reproducibility of the initial calcium level affected our measurements, the relationships between the initial calcium level and the total calcium signal after treatment (as averaged over the interval of 100±200 sec) for all experiments in Figure 1 were statistically analysed and are shown in Figure 2. Typically, the protoplasts of soybean root cells exhibited a pronounced and statistically signi®cant rise in cytosolic [Ca2+] within a minute ((NodBjV (C18:1, MeFuc) in Figure 1a and Figure 2a). The average calcium level following Nod factor application increased by approximately 160% compared to control calcium levels (n = 40, Figure 2a). This rise was sustained for the ®rst 3±5 min, but usually faded during longer incubation (data not shown). By contrast, control addition of acetonitrile/water (50/50) lacking NodBj-V (C18:1, MeFuc) failed to induce a signi®cant change (by t test) in cytosolic [Ca2+] in soybean root cells (n = 40; control in Figure 1a and Figure 2a). Protoplasts from soybean suspension-cultured cells showed a gradual increase in cytosolic [Ca2+] (NodBjV (C18:1, MeFuc) in Figure 1b rather than the rapid response observed in soybean root cell protoplast. The average change in [Ca2+] following the Nod factor application was about 140% (n = 38, Figure 2b); this increase in cytosolic [Ca2+] is also statistically signi®cant (*3 in Figure 2b). The increase returned to basal levels after 10 min (data not shown). The average changes in [Ca2+] following Nod factor solvent application were not signi®cantly different compared to initial [Ca2+] (Figure 2b). The protoplasts of G. soja suspension-cultured cells also showed a pronounced rise of cytosolic [Ca2+] after addition of 10±9 M NodBj-V (C18:1, MeFuc) (Figure 1c and Figure 2c). The average change in [Ca2+] following Nod factor application was about 130% (n = 16, Figure 2c). This increase is also signi®cant (*5 in Figure 2c) and was sustained within the ®rst 4±5 min after the addition of the Nod factor. Again, solvent application without Nod factor did not result in an increased [Ca2+] (n = 16, Figure 2c). Ehrhardt et al. (1996) showed a change in the baseline of the dye signal immediately following Nod factor application in most cells. Gehring et al. (1997) reported that application of NodNGR (Ac, S) at 10±9 M to its homologous legume Vigna unguiculata resulted within seconds in a plateau-like increase in intercellular free calcium in root hairs. Felle et al. (1998) also observed that the Ca2+ ionophore A23187 evoked a rapid depolarization and transient alkalinization of the root hair space that was nearly identical to that observed with NodRm-IV (C16:2, S). Our observations showed that the Nod factor of B. japonicum USDA 110 also induces change in cytosolic [Ca2+] in protoplasts from intact roots as well as microcallus suspension of soybean plants. Ehrhardt et al. (1996) ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 71±78
Nod factor induces transient calcium in¯ux observed an oscillation of the cytosolic calcium level with a mean period of 60 sec that was initiated approximately
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9 min after NodRm-IV (C16:2, S) application to alfalfa root hairs. In this respect, one should note that in our protoplast
Figure 1. Changes in cytosolic [Ca2+] of protoplasts isolated from soybean root cells (a), soybean suspension-cultured cells (b) and wild soybean (Glycine soja) suspension-cultured cells (c) after addition of NodBj-V (C18:1, MeFuc) at 10±9 M (blue curves in the left-hand ®gures), NodBj-V (C18:1) lacking methylfucose (10±8 M, blue curves in the right-hand ®gures), and of an acetonitrile/water mixture (50/50 v/v, red curves, control). In each case, 10 ml of a Nod factor solution in acetonitrile/H2O (50/50) was injected into a 1 ml suspension. The Nod factor concentrations are ®nal concentrations. The arrow indicates the time point of addition.
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suspension-assay system, any asynchronous (cell autonomous) oscillation pattern (if occurring) would be completely average out. The presence of 2-O-methylfucose at the reducing end of NodBj-V has been demonstrated to be essential for eliciting nodulation responses in the host legumes G. max and G. soja (Minami et al., 1996; Stokkerman and
Figure 2. Histogram of the initial calcium level and the total calcium signal after treatment (as averaged over the interval of 100±200 sec) for all experiments in Figure 1. Soybean root cells (a), soybean suspension-cultured cells (b) and wild soybean (Glycine soja) suspension-cultured cells (c) after addition of NodBj-V (C18:1, MeFuc) at 10±9 M, NodBj-V (C18:1) lacking methylfucose at 10±8 M, or an acetonitrile/water mixture (50/50 v/v, control). In each case, 10 ml of a Nod factor solution in acetonitrile/H2O (50/50) was injected into a 1 ml suspension. The Nod factor concentrations are ®nal concentrations. The bars represent standard deviation. White bar, initial calcium level in control; horizontally hatched bar, initial calcium level before Nod factor addition; vertically hatched bar, changes in calcium levels in the control; black bar, NodBj-V (C18:1, MeFuc) addition; diagonally hatched bar, NodBj-V (C18:1) addition. *1, signi®cant (P < 0.001, t test, n = 40); *2, not signi®cant (n = 10); *3, signi®cant (P < 0.001, t test, n = 38); *4, not signi®cant (n = 11); *5, signi®cant (P < 0.001, t test, n = 16); *6, not signi®cant (n = 8).
Peters, 1994). We examined the effect of NodBj-V (C18:1) without methylfucose on the protoplasts of soybean root cells, soybean microcallus suspension cells and wild soybean microcallus suspension cells (NodBj-V(C18:1) as shown in Figure 1a±c and Figure 2a±c). We adjusted the ®nal concentration of the Nod factors to 10±8 M. Those protoplasts showed a de®nite increase in cytosolic [Ca2+] after treatment with NodBj-V (C18:1, MeFuc) (Figure 1a±c). However, NodBj-V (C18:1) without methylfucose induced no signi®cant change in cytosolic [Ca2+] (Figure 1a±c and Figure 2a±c), suggesting that the presence of a methylfucosyl group at the reducing end of the sugar backbone of NodBj-V is essential for inducing increases in cytosolic [Ca2+] in soybean protoplasts. We cannot exclude the possibility that differently decorated Nod factors also give a response in soybean protoplasts. We also tested the effect of NodBj-V (C18:1, MeFuc) on clover (Trifolium pratense) and tobacco BY-2 (Nicotiana tabacum) protoplasts. Suspension culture of clover cells with added Nod factor at 10±9 M did not show any signi®cant changes in cytosolic [Ca2+] (Figure 3a). However, in tobacco BY-2 protoplasts, a decrease in cytosolic [Ca2+] was observed (Figure 3b). The magnitude of this drop is similar to the rise in the soybean culture cells; however, the mechanisms responsible for the drop are unknown. Gehring et al. (1997) reported that root hairs of the non-legume Arabidopsis thaliana did not show an increase in cytosolic [Ca2+] in response to NodNGR (Ac, S). These results suggest that the increase of cytosolic [Ca2+] evoked by NodBj-V (C18:1, MeFuc) is a response speci®c to the cells of the host legumes G. max and G. soja. In all the experiments described above, the incubation media of the protoplasts contained 1 mM Ca2+. There may be two sources of Ca2+ in¯ux into the cytoplasm. One is the intracellular Ca2+ pools postulated to occur in the endoplasmic reticulum and vacuoles (Bush et al., 1989; DuPont et al., 1990), and the other is the extracellular ¯uid. Elimination of Ca2+ from the incubation medium signi®cantly reduced the cytosolic [Ca2+] in the soybean protoplasts dependent on NodBj-V (C18:1, MeFuc) (n = 8, Figure 4a). Furthermore, the addition of an external Ca2+ chelater, 1,2-bis(2-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid (BAPTA), also abolished the Nod factor-induced increase in cytosolic [Ca2+] (n = 10, Figure 4b). These results indicate that the increase in cytosolic [Ca2+] in soybean protoplasts evoked by NodBj-V (C18:1, MeFuc) is caused mainly by transient in¯ux of Ca2+ from the external medium. Figure 4(c) shows the effect of Ca2+ channel inhibitor on the increase in cytosolic [Ca2+] evoked by NodBj-V (C18:1, MeFuc). Pre-treatment of G. max cells with 100 mM verapamil or nitrendipine (data not shown) for 2 min prior to application of NodBj-V (C18:1, MeFuc) also abolished the increase in cytosolic [Ca2+] (n = 12, Figure 4c). In other ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 71±78
Nod factor induces transient calcium in¯ux
Figure 3. Changes in cytosolic [Ca2+] of protoplasts isolated from clover suspension-cultured cells (a) and tobacco BY-2 suspension-cultured cells (b) after addition of NodBj-V (C18:1, MeFuc) at 10±9 M (blue curves) and of an acetonitrile/water mixture (50/50 v/v, red curves, control). In each case, 10 ml of a Nod factor solution in acetonitrile/H2O (50/50) was injected into a 1 ml suspension. The Nod factor concentrations are ®nal concentrations. The arrow indicates the time point of addition.
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Figure 4. Changes in cytosolic [Ca2+] of protoplasts isolated from soybean suspension-cultured cells with calcium-free medium (a), 2 mM BAPTA (b) and 100 mM verapamil (c) after addition of NodBj-V (C18:1, MeFuc) at 10±9 M (blue curves) and of an acetonitrile/water mixture (50/50 v/v, red curves, control). In each case, 10 ml of a Nod factor solution in acetonitrile/H2O (50/50) was injected into a 1 ml suspension. The Nod factor concentrations are ®nal concentrations. The arrow indicates the time point of addition.
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plant studies, verapamil has been shown to be a plasma membrane Ca2+ blocker (Andrejauskas et al., 1985; Graziana et al., 1988; Harvey et al., 1989; Thuleau et al., 1990), with the most likely target being a voltage-gated Ca2+ channel (Freedman et al., 1984; Schumaker and Gizinsk, 1993; Thomine et al., 1994). The fact that verapamil blocks the elevation suggests that voltage-gated calcium channels may be involved, but further analysis is required to substantiate this. Perception of Nod factors by legume plants and the induced signal tranduction pathways leading to the initiation of nodulation processes appear to be very complex. Data from analyses of the responses of legume roots to Nod factors suggest that more than two distinct Nod factor perception systems are involved in the initiation of nodulation processes in corresponding host legumes. One is highly speci®c (Ardourel et al., 1994; Minami et al., 1996), and the other is more or less redundant, because it is thought to be present in nonlegume plants (De Jong et al., 1993). Therefore, to elucidate the signal transduction mechanisms involved in the nodulation process, it is necessary to con®rm the speci®city of the responses of legume cells to speci®c structures of Nod factors. In this paper, we have shown that NodBj-V (C18:1, MeFuc) produced by B. japonicum induces a transient elevation of cytosolic [Ca2+] in protoplasts of root cells and suspension-cultured microcallus cells of soybean plants. This response is apparently speci®c to soybean cells, because it does not increase changes in [Ca2+] in the nonhost legume clover and the non-legume tobacco BY-2. Furthermore, this response is not observed using NodBj-V (C18:1) without the methylfucosyl group, which is inactive on soybean roots. Thus, the results presented in this paper indicate that suspension-cultured cells are useful tools for study of the early steps in signal transduction leading to nodulation.
Experimental procedures Plant cell culture Microcallus suspension-cultures of soybean were made from root primordia of G. max cv. Enrei. They were incubated on B5 medium (Gamborg et al., 1968) containing 0.25% (w/v) Phytagel (Sigma) and 2 mg l±1 2,4-D at 28°C for 4 weeks. Batches of rapidly growing callus tissues were transferred into B5 liquid medium supplemented with 0.1 mg l±1 2,4-D and 0.5 mg l±1 BAP and incubated at 27°C with gentle shaking. A portion of the culture was inoculated to fresh medium every week, and established microcallus suspension cultures were maintained for more than 6 months before experimentation. Suspension cultures of wild soybean (G. soja) were obtained by the same procedures as for soybeans. Suspension cultures of clover (T. pratense) were obtained by the same procedures as for soybeans, except that
the B5 liquid medium contained 0.1 mg l±1 2,4-D and 0.75 mg l±1 BAP. Tobacco suspension-cultured cells of BY-2 were kindly supplied by Dr Y. Ohashi of the National Institute of Agrobiological Resources, Japan.
Preparation of protoplasts Soybean roots of 2±3-day-old seedlings and suspension-cultured cells were incubated in 0.1% w/v Pectolyase Y-23 (Kikkoman Co. Ltd, Chiba, Japan), 3% cellulase Onozuka R-10 (Kinki Yakult, Osaka, Japan) in 0.6 M sorbitol and 15 mM 2-[N-morpholino]ethanesulphonic acid (MES) (pH 5.8) for 2 h at 25°C by gentle rocking. They were ®ltered through a nylon mesh (80 mm), and collected by centrifugation (400 rev min±1 35 g, 3 min, 4°C). The protoplasts were washed three times with buffer A (0.6 M sorbitol, 35 mM sucrose, 6 mM glucose, 1 mM CaCl2 and 15 mM MES, pH 5.8), and ®nally suspended in the same buffer. The protoplasts were kept on ice and used for the experiments within 1 h.
Nod factor puri®cation Nod factors Nod Bj-V (C18:1, MeFuc) and NodBj-V (C18:1) were prepared from cultures of B. japonicum USDA 110 and NAD138 (Stacey et al., 1994), respectively, according to the method described by Stacey (1995). In brief, the bacteria were cultured for 48 h in minimal medium containing soybean seed extracts (Stacey, 1995), and then the whole culture was extracted with nbutanol. The butanol extracts were evaporated to dryness, and then resuspended in acetonitrile/water (82:18, v/v) followed by application to a column (1.5 cm diameter 3 100 cm length) of silica gel 60 (Merck, Darmstadt, Germany) equilibrated with acetonitrile/water (82:18). The column was washed extensively with the same solution, and then the Nod factors were eluted with acetonitrile/water (60:40) from the column. The fractions containing Nod factors were determined by the elution pro®le of authentic NodBj-V (C18:1, MeFuc) and collected. Finally, NodBjV (C18:1, MeFuc) was puri®ed by HPLC with Superpac Pep-S (5 mm, 4 3 250 mm, Pharmacia LKB). Authentic NodBj-V (C18:1) without the methylfucosyl residue and NAD138 strain were kindly supplied by Professor Gary Stacey of the University of Tennessee.
Detection of ¯uorescence emission by spectro¯uorometry The protoplast suspension (approximately 2 ml) was poured into a Petri dish (3 cm diameter) and incubated for 3 h with 10 mM of Fura-PE3 AM (Wako, Tokyo, Japan) on ice with gentle rocking (30 rev min±1, 1.6 g). After Fura-PE3 loading, the protoplasts were pelleted by centrifugation (35 g) and washed three times with icecold buffer A followed by incubation for 30 min at 28°C with gentle rocking. A 1 ml aliquot of the protoplast suspension was transferred into a quartz cell (10 mm path length) at a cell density giving 80% transparency and stirred constantly at 28°C. Cytosolic [Ca2+] in protoplasts was assayed with a luminescence spectrometer (A model LS50B, Perkin Elmer, Foster, UK). Protoplast suspensions were treated with puri®ed NodBj-V (C18:1, MeFuc) or the NodBj-V (C18:1) without methylfucose at various concentrations ranging from 10±9 to 10±8 M or with an equal volume of acetonitrile/water (50:50 v/v) as control. Fluorescence intensity for Fura-PE3 was monitored at an emission wavelength of 510 6 10 nm, with alternating excitation at 340 6 10 and 380 6 10 nm. ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 71±78
Nod factor induces transient calcium in¯ux Auto¯uorescence was measured for protoplasts that had not been loaded with indicator, and this value was subtracted from each ¯uorescence measurement. The percentages of auto¯uorescence in Fura-PE3-loaded cells at both wavelengths were as follows: soybean suspension-cultured cells, 340 nm mean = 2.1%, 380 nm mean = 5.4%; wild soybean suspension-cultured cells, 340 nm mean = 2.0%, 380 nm mean = 5.2%; tobacoo BY-2 cells, 340 nm mean = 2.8%, 380 nm mean = 5.1%. Cell density in a cuvette affected auto¯uorescence levels. Therefore, we checked the cell densities in the cuvettes before testing to maintain a constant concentration. The basal calcium levels varied signi®cantly. We postulated that this variation results from a variation of the cell batch. Therefore, to eliminate this variation, the responses of the two Nod factors were measured for the same batch of cells used to test a negative response (i.e. treatment with the acetonitrile/water mixture (50/50 v/v)); however, we could not eliminate that variation perfectly. In vivo calibration of ratio versus free Ca2+ concentration was performed using Ca2+-EGTA (ethylene glycol-bis(b-aminoethyl ether) N,N,N¢,N¢-tetraacetic acid) buffers to set the free Ca2+ level. Calibration buffers contained 100 mM KCl, 1 mM MgCl2, 10 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulphonic acid) (pH 7.05), 10 mM EGTA and appropriate CaCl2 to give the required free Ca2+ concentration (Gilroy et al., 1989). The dyeloaded protoplast suspensions were incubated for 10 min with these solutions containing 50 mM ionomycin. It was assumed that internal free Ca2+ reached equilibrium with the external Ca2+ level after 10 min of ionomycin treatment. Calibration data were obtained at the end of each study.
Acknowledgements We thank Professor Gary Stacey (University of Tennessee, Knoxville, USA) for providing NodBj-V (C18:1, MeFuc) and NodBj-V (C18:1). We also thank Dr Eva Kondorosi (Institute des Sciences Vegetales, CNRS, France), Dr Duncan A. Vaughan (National Institute of Agrobiological Resources, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Japan) and Professor Robert W. Ridge (Department of Biology, International Christian University, Tokyo, Japan) for revising this manuscript. This work was supported by grants from both the Bio-Design Project of the Ministry of Agriculture, Forestry and Fisheries, Japan, and the Asian Network on Microbial Researches of the Science and Technology Agency, Japan.
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