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Keywords:

  • elicitor;
  • Lipid A;
  • lipopolysaccharides;
  • Medicago;
  • Nicotiana;
  • oxidative burst;
  • plant defence;
  • Sinorhizobium meliloti

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Medicago sativa (alfalfa), Medicago truncatula and Nicotiana tabacum cell suspension cultures, responding to elicitation with the production of reactive oxygen species (ROS), were used to analyse the suppressor (and elicitor) activity of lipopolysaccharides (LPS) of the symbiotic soil bacterium Sinorhizobium meliloti.
  • • 
    In order to identify the epitopes of the LPS molecule recognized by the plant, S. meliloti mutants defective in LPS biosynthesis and hydrolytically obtained Lipid A were analysed for biological activity.
  • • 
    Lipopolysaccharides isolated from Sinorhizobium meliloti mutants 6963 (altered core region) and L994 (no long-chain fatty acid) showed the same ability to suppress the oxidative burst in host plant cell cultures as the wild-type LPS. Lipid A also displayed the same suppressor activity. By contrast, rhizobial LPS, but not Lipid A, was active as an inducer of the oxidative burst reaction in cell cultures of the nonhost Nicotiana tabacum.
  • • 
    In host plants of Sinorhizobium meliloti the Lipid A part is sufficient to suppress the oxidative burst, but in non-host plants at least some sugars of the LPS core region are required to induce defence reactions.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The soil bacterium Sinorhizobium meliloti is able to establish a nitrogen-fixing symbiosis with its host plants alfalfa (Medicago sativa) and the model legume Medicago truncatula. This symbiotic interaction leads to the formation of a completely new organ, the so-called root nodule that hosts the rhizobia intracellularly. Inside the nodules, they are able to fix atmospheric nitrogen and convert it to ammonia, which is then provided to the plant (Hirsch, 1992).

Since plants encounter not only beneficial microbes, but also have to cope with phytopathogenic microorganisms, they have developed a set of defence mechanisms that enables them to identify pathogens by so-called PAMP (pathogen-associated molecular patterns) and start suitable defence reactions, such as the induction of a hypersensitive response (HR) or the production of reactive oxygen species, as it was lately reviewed by Montesano et al. (2003). By contrast, in symbiosis, a complex molecular dialogue between the two interacting partners regulates plant defence reactions and thus enables symbiosis (Djordjevic et al., 1987).

In addition to plant flavonoids (Brewin, 1991) and rhizobial nodulation factors (Roche et al., 1991), bacterial surface saccharides such as exopolysaccharides (EPS) and lipopolysaccharides (LPS) form another important class of signal molecules for an effective symbiosis (Niehaus & Becker, 1998). These molecules are most likely involved in the infection process during establishment of the symbiosis. An indication of the significance of LPS as a signal molecule could be deduced from bacterial mutants defective in LPS biosynthesis. Various rhizobial mutants with alterations in LPS were defective in symbiotic interactions with their host plants (Carlson et al., 1987; Lagares et al., 1992; Campbell et al., 2003). lpsB mutants of S. meliloti exhibit an altered LPS and fail to establish an effective symbiosis with M. truncatula (Niehaus et al., 1998). The symbiotic defect of the lpsB mutant was correlated with typical indications for plant defence reactions like the production of reactive oxygen species. By contrast, on M. sativa an effective symbiosis was established by this mutant, indicating a different specific recognition of the LPS by alfalfa than by M. truncatula. Recently, the role of rhizobial LPS in symbiosis was partly characterized by Albus et al. (2001), who showed that the isolated LPS was able to suppress the elicitor-induced oxidative burst in cell suspension cultures of the host plant M. sativa. Interestingly, in the nonhost plant Nicotiana tabacum (tobacco) they exhibit an opposite effect and elicit the plant pathogen defence comparable to that of LPS isolated from the phytopathogen Xanthomonas campestris pv. campestris (S. Braun and A. Meyer, pers. comm.).

In this work, we examined in more detail, which parts of the S. meliloti lipopolysaccharides are recognized by host and nonhost plants. For this purpose, we compared the suppressor, and elicitor activity respectively, of the wild-type LPS with a core- (Sm6963) (Lagares et al., 1992) and a Lipid A-mutant (acpXL) (Sharypova et al., 2003) and pure Lipid A obtained by hydrolysis of wild-type LPS after elicitation in M. sativa, M. truncatula and N. tabacum cell cultures.

By using LPS of S. meliloti mutants and chemically obtained substructures the biological active part of the molecule could be defined in M. sativa and M. truncatula cell cultures.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chemicals and materials

Chemicals were purchased from ICN Biomedicals, Inc. (Eschwege, Germany) or Sigma (Taufkirchen, Germany), unless noted otherwise.

Bacterial strains and culture conditions

In this work, the S. meliloti wild-type strain 2011 (Casse et al., 1979) and the LPS mutants 6963 (Lagares et al., 1992) and L994 (Sharypova et al., 2003) were used. For the isolation of lipopolysaccharides the cells were grown on tryptone yeast (TY)-agar plates supplemented with 0.4% glucose (w : v) at 28°C.

Cell suspension cultures and plant material

Medicago truncatula‘Jemalong’ cell suspension cultures were obtained by placing sterile root explants of 1-wk-old seedlings on sterile MS medium (Murashige & Skoog, 1962) supplemented with the phytohormones 2,4-dichlorophenoxyacetic acid (1 mg l−1) and kinetin (0.1 mg l−1).

Eight-week-old explants frequently produced callus tissue and nonnecrotic calli were subcultivated to establish callus cell cultures of M. truncatula root tissue. Cell suspension cultures were then obtained by transferring small amounts of callus tissue into liquid MS medium and agitating on a shaker. Cell cultures consisting of single cells and small aggregates of cells were used for subcultivation.

Medicago truncatula‘Jemalong’, M. sativa‘Du Puits’ and N. tabacum cell suspension cultures were maintained in MS medium (Murashige & Skoog, 1962) and subcultured every 7 d as described by Baier et al. (1999).

Hot phenol extraction and purification of LPS

Cells were grown on TY agar plates for 3 d and washed from the plates with 0.9% NaCl (w : v). After centrifugation at 5000 g for 20 min, cell pellets of approx. 60 g wet weight were resuspended in H2O and LPS was extracted using the hot phenol–water method according to Westphal & Jann (1965). The water phase was dialysed extensively against water, proteins and nucleic acids were removed by treatment of the dialysate with 100 µg ml−1 DNAse I (Boehringer Mannheim, Germany), 15 µg ml−1 RNAse (Boehringer Mannheim) and 150 µg ml−1 proteinase K, re-dialysed and lyophilized. The LPS was resuspended and purified by ultracentrifugation at 100 000 g. Subsequently, the LPS was further purified by gel-permeation chromatography using a Sephadex G-50 matrix in a pyridine acetate solvent (pyridine 0.4%, acetic acid 1%).

Generation of Lipid A from rhizobial LPS

The Lipid A was released from the complete lipopolysaccharides by mild acid hydrolysis (1% acetic acid at 100°C for 1 h) and isolated by ultracentrifugation at 100 000 g. For oxidative burst measurements Lipid A was diluted to 5 mg ml−1 in dimethyl sulphoxide (DMSO).

Determination of the oxidative burst reaction in plant cell suspension cultures

The detection of the oxidative burst was performed using the H2O2-dependent chemiluminescence reaction described by Warm & Laties (1982). Three to five days after subcultivation 2 g of cell material from the cell suspension cultures were diluted in 8 ml of preincubation medium (3% w : v sucrose in 0.04× MS; Murashige & Skoog, 1962) and incubated for 3–4 h. For the measurement of the oxidative burst 200 µl aliquots of these suspensions were mixed with 700 µl phosphate buffer (50 mm K-phosphate; pH 7.9) and 100 µl 1.2 mm luminol in the same phosphate buffer. The reaction was started by the addition of 100 µl of 14 mm potassium-hexacyanate. The luminescence was measured with a Sirius Luminometer from Berthold Detection Systems (Pforzheim, Germany).

Invertase was used as a general elicitor of plant pathogen defence with a final concentration of 20 µg ml−1, but in contrast to the work of Fath & Boller (1996) the complete enzyme was added to the cell cultures and was able to stimulate the production of reactive oxygen species.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolated lipopolysaccharides of the S. meliloti mutants 6963 and L994 displayed wild-type suppressor activity in cell cultures of M. sativa and the model legume M. truncatula

To characterize which parts of the rhizobial LPS are recognized by host plants, the purified LPS from S. meliloti Rm 2011 and the mutants 6963 (lpsB) and L994 (acpXL) were tested in cell suspension cultures of M. sativa and M. truncatula for their ability to influence the elicitor-induced oxidative burst reaction (Baier et al., 1999; Albus et al., 2001).

Lipopolysaccharides isolated from the wild-type S. meliloti 2011 are able to suppress the elicitor induced oxidative burst in cell cultures of the host plant M. sativa (Albus et al., 2001). Since the LPS-deficient mutant 6963 is able to establish an effective symbiosis with M. sativa but fails to do so on M. truncatula, a cell suspension culture of this legume was established from seeds of the cultivar Jemalong. Sterile seeds were germinated and explants of the seedlings then cultivated on solid MS plant medium. The resulting callus tissue was transferred to liquid MS medium and selected for growth of single cells and small cell aggregates. The resulting cell suspension culture was also selected for responsiveness to invertase using the oxidative burst assay.

The time-dependent production of H2O2 upon application of invertase, here used as a general elicitor (Fath & Boller, 1996), was determined as described in the Materials and Methods section. The addition of invertase to cell suspension cultures of M. sativa and M. truncatula to a final concentration of 20 µg ml−1 lead to a strong oxidative burst with a maximum hydrogen peroxide concentration of 5–8 µm H2O2 20–25 min after elicitation (Fig. 1). The simultaneous application of invertase and wild-type LPS or S. meliloti 6963 LPS to a final concentration of 5 µg ml−1 suppressed the production of H2O2 almost completely (Fig. 1a; Table 1) in M. sativa cell suspension cultures. In the M. truncatula cell suspension cultures both LPS species (wild-type and 6963) were able to suppress the elicitor induced oxidative burst, but the suppression level (Fig. 1b) was somewhat lower (30.8 ± 12%, SD with five replicates in the case of 6963 LPS) compared with M. sativa (Table 1).

image

Figure 1. Suppression of the invertase induced oxidative burst reaction in Medicago sativa and Medicago truncatula cell suspension cultures by isolated lipopolysaccharides (LPS) from Sinorhizobium meliloti mutants 6963 and L994. The hydrogen peroxide generation in M. sativa (a,c) and M. truncatula (b,d) was analysed after application of invertase with or without LPS (wild-type, 6963 and L994); 20 µg ml−1 invertase (closed squares), 5 µg ml−1 wild-type LPS in combination with 20 µg ml−1 invertase (triangles), 5 µg ml−1 6963 LPS in combination with 20 µg ml−1 invertase (open circles) and 5 µg ml−1 L994 LPS with 20 µg ml−1 invertase (open squares) was applied to the culture. The same amount of pure water was added (diamonds) as a negative control. The H2O2 concentration in the culture was measured using the luminol luminescence assay and is given in µm H2O2. One representative experiment from several replicates is shown.

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Table 1.  Suppressor activities of Sinorhizobium meliloti wild-type lipopolysaccharides (LPS), mutant (6963 and L994) LPS and wild-type Lipid A in suspension cell cultures of the host plants Medicago sativa and Medicago truncatula
Suppressor% Suppression of the oxidative burst
M. sativaM. truncatula
  1. An oxidative burst was induced in the cell cultures by the general elicitor invertase with final concentration of 20 µg ml−1. For suppression, LPS and Lipid A were applied simultaneous with the invertase to a final concentration of 5 µg ml−1. The suppression is given as reduction in H2O2 production as percentage compared with invertase alone (100%). This type of representation was chosen because there is great variation in total H2O2 production between different measurements but only little variation in the suppression levels.

S. meliloti 2011 LPS73.4 ± 9.4  41 ± 4.2
S. meliloti 6963 LPS75.5 ± 4.930.8 ± 12
S. meliloti L994 LPS63.8 ± 17.840.6 ± 12.9
S. meliloti Lipid A64.9 ± 7.648.7 ± 22.7

Under the same conditions L994 LPS also showed wild-type suppressor activity (Fig. 1c,d). The level of suppression reached approx. 70% in M. sativa cell cultures. In M. truncatula a much lower suppression of the oxidative burst (40.6 ± 12.9%, SD with 6 replicates) was monitored (Table 1). These observations indicated that the biologically active and plant-recognized part of the LPS molecule is not modified in the S. meliloti 6963 mutant. The most noticeable feature of the rhizobial LPS, the hydroxylated C28 fatty acid, is also not necessary for the suppression of the oxidative burst in host plant cells.

The suppressor activity resides in the Lipid A part of the LPS molecule

Since LPS isolated from S. meliloti mutants was still suppressor active, the most basic structure, the Lipid A, was tested for biological activity.

The Lipid A was chemically released from LPS by mild acid hydrolysis with acetic acid and subsequent ultracentrifugation. DMSO was used as solvent to prevent micelle formation leading to a maximum DMSO concentration in the plant cell cultures of c. 1%. M. sativa and M. truncatula cell suspension cultures showed no increased or reduced H2O2 production after addition of the solvent (data not shown). In this experiment, the invertase induced an oxidative burst in alfalfa with a maximum hydrogen peroxide concentration of 7 µm after 20 min, followed by a steady decline (Fig. 2). The simultaneous addition of rhizobial Lipid A to 5 µg ml−1 reduced the H2O2 production to the same level as the complete LPS (Table 1). In M. truncatula cell suspension cultures rhizobial Lipid A and LPS showed also suppressor activity, but the degree of suppression was only c. 50% (48.3 ± 22.7%; SD with five replicates). These observations indicate that the core and O-antigenic part of the LPS molecule is not necessary for the perception of these signal molecules by host plants in order to suppress the oxidative burst.

image

Figure 2. Suppression of the invertase-induced oxidative burst reaction in Medicago sativa and Medicago truncatula cell suspension cultures by chemically released Lipid A from Sinorhizobium meliloti lipopolysaccharides (LPS). The H2O2 generation was monitored after application of invertase with and without addition of LPS and Lipid A to M. sativa (a) and M. truncatula (b) cell suspension cultures; 20 µg ml−1 invertase (squares), 5 µg ml−1 wild-type LPS in combination with 20 µg ml−1 invertase (triangles) and 5 µg ml−1 chemically released rhizobial Lipid A plus 20 µg ml−1 invertase (circles) were added to the cell suspension cultures. The H2O2 concentration in the culture was measured using the luminol luminescence assay and is given in µm H2O2. One representative experiment from several replicates is shown.

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In cell cultures of the nonhost plant Nicotiana tabacum complete rhizobial LPS, but not Lipid A, act as an elicitor of the oxidative burst reaction

Lipopolysaccharides of Gram-negative bacteria are able to induce and modulate defence reactions in plants (Dow et al., 2000; Coventry & Dubery, 2001; Meyer et al., 2001). The structural requirements for LPS for the prevention of a hypersensitive response (HR) have been investigated in several pathogenic plant–microbe interactions (Graham et al., 1977) and it was shown that the minimal structure comprises Lipid A, 3-deoxy-D-manno-octulosonic acid (KDO) and additional heptoses (Newman et al., 1997) of the Salmonella minnesota LPS. Considering this, it was even more surprising that the Lipid A substructure of the rhizobial LPS kept its suppressor activity in host plants after acid hydrolysis. To test whether this is a general feature of rhizobial LPS, the effects of LPS and Lipid A in cell suspension cultures of the nonhost plant N. tabacum were analysed.

The addition of invertase to the cell culture to a final concentration of 20 µg ml−1 led to an oxidative burst reaction with a maximum of 33 µm H2O2 25 min after elicitation (Fig. 3). Thereafter, the H2O2 concentration declined steadily and reached base level after 1 h. As can be seen in the negative control, DMSO (final concentration of c. 1%) had no effect on hydrogen peroxide production in tobacco.

image

Figure 3. Elicitation of the oxidative burst reaction in cell cultures of Nicotiana tabacum with Sinorhizobium meliloti lipopolysaccharides and Lipid A. Generation of hydrogen peroxide in cell suspension cultures of the nonhost tobacco induced by 20 µg ml−1 invertase (squares), 20 µg ml−1 rhizobial lipopolysaccharides (circles) and 20 µg ml−1 chemically released rhizobial Lipid A diluted in dimethyl sulphoxide (DMSO) (triangles). DMSO was used as a negative control (diamonds). The H2O2 concentration in the culture was measured using the luminol luminescence and is given in µm H2O2. One representative experiment from several replicates is shown.

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A 20 min delayed oxidative burst was induced by 20 µg ml−1 rhizobial LPS, compared with the invertase, which reached its maximum after 45 min. The LPS burst remained at maximum level for c. 35 min with a slow subsequent decline to 20 µm H2O2 after 2 h. The same concentration of Lipid A was unable to induce any detectable oxidative burst and showed the same H2O2 concentration as the negative control (Fig. 3). In additional experiments it was shown that the simultaneous application of rhizobial Lipid A and Invertase to the tobacco cell culture was unable to modify the oxidative burst produced by Invertase alone (data not shown).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

During the establishment of the root nodule symbiosis a massive infection of the host plant takes place. In the case of infection by wild-type rhizobia, no obvious signs of plant defence are visible. A closer look at the curled root hair and the initiation of the infection thread revealed a moderate oxidative burst at the primary site of infection (Santos et al., 2001). In addition, excessive infection of the host plant is prevented by defence-like abortion of infection threads inside the root hair (Vasse et al., 1993). Obviously, plant defence reactions are involved in the early steps of this beneficial plant–microbe interaction. Plant mutants that defeat an infection by rhizobia (Mitra & Long, 2004) and rhizobial mutants defective in EPS (Niehaus et al., 1993) or in LPS (Niehaus & Becker, 1998) biosynthesis induce the plant defence system. These findings indicate that an active suppression of plant defence could take place during the establishment of this symbiosis. Evidence supporting this concept came from the observation that isolated S. meliloti lipopolysaccharides were able to suppress an elicitor-induced oxidative burst in cell cultures of M. sativa, but not in nonhost cell cultures of N. tabacum (Albus et al., 2001).

To further characterize the suppressor activity of the S. meliloti LPS, a cell suspension culture of the model legume M. truncatula was established and selected for responsiveness against the general elicitor invertase (Fath & Boller, 1996). Simultaneous application of invertase and S. meliloti 2011 wild-type LPS led to the production of less hydrogen peroxide in the M. truncatula cell culture than application of invertase alone. Interestingly, the general level of suppressor activity of the S. meliloti LPS was much lower compared with the M. sativa cell culture. One explanation for these results may be that M. truncatula is a Mediterranean legume and therefore not a perfect host plant that S. meliloti strain 2011 encounters in its natural environment. This would indicate that the biologically active LPS contributes to the host-specificity, even though it was published that the S. meliloti LPS carries not enough structural information to be strain-specific (Reuhs et al., 1998).

The next step in characterizing the biological activity of the S. meliloti LPS was the identification of the suppressor-active part of the LPS molecule. Two S. meliloti LPS mutants (6963 and L994) were examined for suppressor activity in cell suspension cultures of M. sativa and M. truncatula. Mutant 6963 carries a defect in the lpsB gene and produces LPS with a (probably) changed core region (Lagares et al., 1992; Kanipes et al., 2003) that generates a delayed Fix+ phenotype in M. sativa and a Fix phenotype in M. truncatula (Niehaus et al., 1998). Isolated LPS from this mutant acted as a suppressor in M. sativa and in M. truncatula cell suspension cultures. The Fix+ phenotype on M. sativa and the Fix phenotype on M. truncatula (Niehaus et al., 1998) is not as clearly reflected on the cell culture level. Nevertheless, the suppressor effect was always weaker in M. truncatula cell cultures compared with M. sativa, as was reported earlier in this study for the wild-type LPS.

From this observation two functions can be proposed for the rhizobial LPS: one early function, most probably during the infection of the root hairs, suppressing the oxidative burst, and a late function involved in the endocytotic uptake of rhizobia in the infected nodule cells.

The same suppressor activity was obtained for the LPS from S. meliloti mutant L994, which carries a mutation in the acpXL gene. This leads to the loss of the long-chain fatty acid in the lipid part of the LPS and induces a delayed Fix+ phenotype in symbiosis (Sharypova et al., 2003). Lipopolysaccharides of the mutant L994 displayed the same level of suppression in cell cultures of M. sativa and M. truncatula as wild-type LPS. This result is interesting, because it implies that the most noticeable characteristic feature of rhizobial LPS, the hydroxylated C28 fatty acid (Hollingsworth & Carlson, 1989; Bhat et al., 1994), is not necessary for the recognition of the LPS by host plants in order to suppress the production of reactive oxygen species. It is possible that the delayed nodulation phenotype results from a beneficial effect of the C28 fatty acid in later stages of symbiosis, such as the release of the bacteria from the infection thread or uptake into the plant cells, or in the establishment of functional symbiotic membranes, as reported previously for the acpXL mutant of Rhizobium leguminosarum in pea (Vedam et al., 2004).

Since both mutants analysed so far in this study did not show differences in their ability to suppress the oxidative burst reaction in the host plants M. sativa and M. truncatula, the most basic form of LPS, Lipid A, was used for further experiments. The chemically released Lipid A moiety of the LPS was also able to suppress the production of reactive oxygen species with the same efficiency as the complete LPS in both Medicago cell cultures tested. A solvent (DMSO) was necessary to dilute the hydrophobic Lipid A and to increase the bioavailability of the molecule. To date, this recognition of bacterial Lipid A has only been known for endotoxin in the animal immune system (Galanos et al., 1986; Ulmer et al., 2002).

It would be interesting for future studies to compare the suppressor activity of LPS and Lipid A of S. meliloti with other LPS and Lipid A species from the Rhizobiaceae and other, non-symbiotic Enterobacteriaceae such as Escherichia coli.

To elucidate, whether the structural features recognized by the host plants are specific for the rhizobia-legume symbiosis, the effects of LPS and Lipid A (Fig. 4) from S. meliloti were tested for elicitor activity in cell suspension cultures of N. tabacum. The isolated entire lipopolysaccharides induced a potent and long-lasting oxidative burst in the cell cultures as reported by Albus et al. (2001), but the addition of Lipid A did not result in the formation of extra hydrogen peroxide. Obviously, Lipid A represents no elicitor-active structure. This finding also implies that at least a part of the carbohydrate LPS substructure is necessary for elicitation.

image

Figure 4. Chemical structure of the Sinorhizobium meliloti Lipid A with additional 3-deoxy-d-manno-2-octulosonic acid (KDO) residues. Shown here is the structure of the S. meliloti Lipid A as it was published recently. It consists of two glucosamines, two phosphates, five fatty acids and a hydroxy-butyrate. In this image two additional KDO residues were added to the Lipid A structure (Kanipes et al., 2003).

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For plants, it is of vital importance to recognize invading pathogens in order to start suitable defence reactions, but at some point in evolution it became more beneficial to host invading nitrogen-fixing bacteria instead of defending against them (Sharifi, 1983; Quispel, 1998). This hypothesis is substantiated by the finding that M. truncatula expresses two symbiosis-specific nodulins that share homologies to pathogenicity-related genes (Gamas et al., 1998). In this work, it was shown that even the basic Lipid A structure shows the same biological activity in host plant cell cultures as the LPS, but was completely inactive in the non-host plant tobacco. These results indicate a clear difference in the recognition of LPS in pathogenicity and symbiosis in plants, generating an even more complex picture of LPS perception and signalling in plants than already suggested. In this background, it becomes more comprehensible that M. sativa and M. truncatula might possess two different perception systems for LPS: one for the recognition of pathogenic Gram-negative bacteria and another for the symbiosis-specific recognition of rhizobia.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We are very grateful to Laryssa Sharypova for providing the S. meliloti acpXL mutant. This work was supported financially by the Special Collaborative Project 549 (SFB 549) from the German Research Council (DFG).

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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