Medicago-Sinorhizobium-Ralstonia: A Model System to Investigate Pathogen-Triggered Inhibition of Nodulation
- Claire Benezech †
- Alexandre Le Scornet
- Benjamin Gourion
- LIPM, Université de Toulouse, INRA, CNRS, 84195 Castanet-Tolosan, France
How plants deal with beneficial and pathogenic microorganisms and how they can tolerate beneficial ones and face pathogens at the same time are questions that remain puzzling to plant biologists. Legume plants are good models to explore those issues, as their interactions with nitrogen-fixing bacteria called rhizobia results in a drastic and easy-to-follow phenotype of nodulation. Intriguingly, despite massive and chronic infection, legume defense reactions are essentially suppressed during the whole symbiotic process, raising a question about a potential negative effect of plant immune responses on the establishment of nodulation. In the present study, we used the model legume, Medicago truncatula, coinoculated with mutualistic and phytopathogenic bacteria, Sinorhizobium medicae and Ralstonia solanacearum, respectively. We show that the presence of R. solanacearum drastically inhibits the nodulation process. The type III secretion system of R. solanacearum, which is important for the inhibition of pathogen-associated molecular pattern–triggered immunity (PTI), strongly contributes to inhibit nodulation. Thus, our results question the negative effect of PTI on nodulation. By including a pathogenic bacterium in the interaction system, our study provides a new angle to address the influence of the biotic environment on the nodulation process.
Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Legumes can interact with nitrogen-fixing bacteria called rhizobia, which provide them nitrogen. These bacteria are hosted in root nodules. At the early stages of the interaction, defense reactions that are normally induced by microbes through the plant perception of microbial-associated molecular patterns (MAMPs) or effectors are suppressed or weak and transient. For instance, in the model legume Medicago truncatula, defense-related genes are transitorily upregulated upon interaction with Sinorhizobium spp. (Lohar et al. 2006), but then, there are no typical defense reactions all along the interaction (Benezech et al. 2020b). Interestingly, these transient defense responses are strongly increased when Medicago spp. interact with a bacterial mutant altered in production of exopolysaccharide (EPS), a determinant known to suppress MAMP-triggered immunity (MTI) (Aslam et al. 2008; Jones et al. 2008). These observations raise a question about the influence of the biotic environment, including pathogens, on nodulation. In the past, this question was mostly addressed using systems devoid of pathogens, in which the presence of the aggressor was mimicked by treatment with MAMP (Lopez-Gomez et al. 2012) or in which the plant defense signaling pathways were artificially switched off (Stacey et al. 2006). Those molecular studies showed that plant defenses have an antagonistic effect on nodulation. However, other studies indicate an overlap of the plant response to MAMP and to nodulation signals (Serna-Sanz et al. 2011). Furthermore, boosting MTI through the constitutive expression of EFR, the receptor of the elf26 MAMP shared by bacterial pathogens and rhizobia (Berrabah et al. 2019), has no strong impact on nodulation (Pfeilmeier et al. 2019).
To evaluate whether the pathogen impacts the nodulation process, an in-vitro tripartite system involving M. truncatula (Pecrix et al. 2018), its symbiotic partner Sinorhizobium medicae WSM419 (Reeve et al. 2010), and the phytopathogenic bacterium Ralstonia solanacearum GMI1000 (Salanoubat et al. 2002) was set up. R. solanacearum has been known to trigger wilting symptoms on a non-nodulated M. truncatula A17 ecotype that is susceptible to R. solanacearum GMI1000 (Turner et al. 2009; Vailleau et al. 2007). R. solanacearum GMI1000 is a model soil pathogen that colonizes the xylem of its hosts. It displays a T3SS that allows the translocation of effector proteins (T3SEs) into the cells of its host. A total of 24 Ralstonia T3SEs were shown to inhibit MTI in Arabidopsis thaliana and in Nicotiana benthamiana (Landry et al. 2020), although this role has not yet been formally described in genus Medicago, in which GMI1000 effectors are crucial for pathogenicity (Vailleau et al. 2007). When efficient, this suppression of immunity is referred to as effector-triggered susceptibility (ETS) (Jones and Dangl 2006). On M. truncatula A17, despite development of plant defenses, R. solanacearum colonizes the nodules and roots (Benezech et al. 2020a), but the effect of this pathogen on M. truncatula nodulation has so-far not been described.
In our laboratory, the routine conditions used to study R. solanacearum–M. truncatula pathogenic interaction and nodulation differ only by temperature (Fahraeus solid medium [Fåhraeus 1957] at 28°C for interactions with R. solanacearum and at 25°C for nodulation). To determine whether temperatures adapted for pathogenic interaction might be used to evaluate the impact of R. solanacearum on nodulation, we first verified whether M. truncatula can nodulate at 28°C (Fig. 1). At both 14 and 21 days postinoculation (dpi), plants cultivated at 28°C display an average of one nodule per plant, while 25°C controls harbor two times as many nodules. These results indicate that, despite a negative impact of high temperature on the symbiotic process, 28°C is a temperature suitable to study the effect of R. solanacearum on Medicago nodulation.
Thus, M. truncatula seedlings were then coinoculated with both the pathogenic and the mutualistic bacteria at equal densities onto plants (optical density at 600 nm [OD600] = 0.1 in sterile distilled water), and the development of nodules was monitored for 28 days. S. medicae and R. solanacearum cells were cultivated overnight in, respectively, tryptone yeast (Beringer 1974) and Phi medium (Poueymiro et al. 2009). While on control plants, inoculated only with the rhizobium, nodules were observable from day 14 and continued to develop continuously until the last point analyzed (28 dpi) (Fig. 2A), we were not able to detect any nodules on coinoculated plants during the whole experiment (Fig. 2B). Plant age and nutritional status can affect, respectively, plant susceptibility to pathogens and interaction with rhizobia (Campisi and Robert 2014). For this reason, we also analyzed the influence of R. solanacearum on nodulation with older plants in which the nitrogen storage in cotyledons was already reduced (11-day-old seedlings). No nodules were formed in those coinoculated plants (Fig. 2D). In contrast, in seedlings inoculated only with S. medicae, the nodule number increased over the time (Fig. 2D). At 28 dpi, for plants that were inoculated when they were 0 and 11 days old, two nodules per plant were present in average on 34 control plants, but none were detected on 34 coinoculated plants (Fig. 2D). These results indicate that the presence of the phytopathogenic bacterium interferes drastically and early with nodulation (independently of the nitrogen need of the plants).
To evaluate whether R. solanacearum–mediated inhibition of nodulation is dose-dependent and potentially due to direct antagonism with S. medicae or to root congestion by R. solanacearum, the same experiment was performed with ten times fewer cells of R. solanacearum. In such a condition, as when the two microorganisms were inoculated at equal densities, no nodules developed on coinoculated plants (Fig. 2D), suggesting that nodulation inhibition is not due to competition for the access to the infection site.
Because of its key role in the subversion of plant cell physiology, we evaluated the importance of R. solanacearum T3SS in the inhibition of nodulation. Seedlings were coinoculated with S. medicae and the hrcV mutant of R. solanacearum, deficient for the T3SS (Cunnac et al. 2004). Root tip browning was observed on seedlings coinoculated with S. medicae and the R. solanacearum wild-type strain (WT), which can be due to the appearance of disease (ETS) or the development of defense responses (effector-triggered immunity–like) (Fig. 2E). In contrast, no such a symptom was detectable when plants were coinoculated with S. medicae and the hrcV mutant (Fig. 2F). On those plants, nodules were frequently observed (Fig. 2C). However, the Ralstonia T3SS mutant still has the ability to partially inhibit nodulation by S. medicae (Fig. 2D). The effect was not significantly different when the disarmed pathogen was inoculated at reduced density and a similar tendency was observed for plants inoculated immediately after transfer on Fahreus medium. Indeed, from 28 dpi, regardless the age of seedlings and the densities of R. solanacearum inoculum (OD600 = 0.1 or 0.01), on average, less than one nodule per plant was present on 34 coinoculated plants (Fig. 2D). Thus, our results indicate that R. solanacearum T3SS strongly contributes to the inhibition of nodulation.
Our data reinforces the idea that the biotic environment can impact the nodulation process. Legume-rhizobia interactions are most often studied with experimental systems restricted to the two symbiotic partners. However, such a reductionist approach is far from the complex reality of natural environments and it is important to develop experimental systems including one or more additional microbial partners to understand the complexity of the interactions. We took a first step in this direction that allows evaluating how other interactions can influence nodulation efficiency. We observed that WT R. solanacearum totally inhibits nodulation, perhaps through defense response elicitation (although A17 is susceptible to GMI1000). In contrast, the hrcV mutant does not totally block nodulation (Fig. 3B). This partial inhibition of nodulation might be due to induction of defense responses that are not counteracted by an effector normally translocated through R. solanacearum T3SS. Among the factors potentially triggering defense responses are, notably, surface components acting as MAMPs and ethylene produced by Ralstonia spp. Indeed, ethylene is both a defense response elicitor and an inhibitor of nodulation in Medicago spp. (Oldroyd et al. 2001; Peters and Crist-Estes 1989; Valls et al. 2006). Other R. solanacearum factors as well as rhizobial determinants, such as the nod factors, EPS, and 1-aminocyclopropane-1-carboxylate deaminase (that might reduce ethylene production by degrading the ethylene precursor), could contribute to the suppression of defense responses induced by the disarmed pathogen. In addition, WSM419 strain possesses a T4SS that might also play such a role (Fig. 3C). Whether the reduced inhibitory effect of the hrcV mutant on nodulation reflects an inability of PTI to prevent nodule development in our system or whether the rhizobium or the hrcV mutant are able to suppress PTI induced by the R. solanacearum mutant remains to be determined (Fig. 3).
The author(s) declare no conflict of interest.
- 2008. Bacterial polysaccharides suppress induced innate immunity by calcium chelation. Curr. Biol. 18:1078-1083. https://doi.org/10.1016/j.cub.2008.06.061 Crossref, Medline, ISI, Google Scholar
- 2020a. Medicago-Sinorhizobium-Ralstonia co-infection reveals legume nodules as pathogen confined infection sites developing weak defenses. Curr. Biol. 30:351-358.e4. https://doi.org/10.1016/j.cub.2019.11.066 Crossref, Medline, ISI, Google Scholar
- 2020b. Legumes tolerance to rhizobia is not always observed and not always deserved. Cell. Microbiol. 22:e13124. https://doi.org/10.1111/cmi.13124 Crossref, Medline, ISI, Google Scholar
- 1974. R factor transfer in Rhizobium leguminosarum. Microbiology 84:188-198. https://doi.org/10.1099/00221287-84-1-188 Crossref, ISI, Google Scholar
- 2019. Legume nodules: Massive infection in the absence of defense induction. Mol. Plant-Microbe Interact. 32:35-44. https://doi.org/10.1094/MPMI-07-18-0205-FI Link, ISI, Google Scholar
Cell senescence: role in aging and age-related diseases. Pages 45-61 in: Interdisciplinary Topics in Gerontology. L. Robert, and T. Fulop, eds. S. Karger AG, Basel. Google Scholar
- 2004. Inventory and functional analysis of the large Hrp regulon in Ralstonia solanacearum: Identification of novel effector proteins translocated to plant host cells through the type III secretion system. Mol. Microbiol. 53:115-128. https://doi.org/10.1111/j.1365-2958.2004.04118.x Crossref, Medline, ISI, Google Scholar
- 1957. The Infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. Microbiology 16:374-381. https://doi.org/10.1099/00221287-16-2-374 Crossref, ISI, Google Scholar
- 2006. The plant immune system. Nature 444:323-329. https://doi.org/10.1038/nature05286 Crossref, Medline, ISI, Google Scholar
- 2008. Differential response of the plant Medicago truncatula to its symbiont Sinorhizobium meliloti or an exopolysaccharide-deficient mutant. Proc. Natl. Acad. Sci. 105:704-709. https://doi.org/10.1073/pnas.0709338105 Crossref, Medline, ISI, Google Scholar
- 2020. The large, diverse, and robust arsenal of Ralstonia solanacearum type III effectors and their in planta functions. Mol. Plant Pathol. 21:1377-1388. https://doi.org/10.1111/mpp.12977 Crossref, Medline, ISI, Google Scholar
- 2006. Transcript analysis of early nodulation events in Medicago truncatula. Plant Physiol. 140:221-234. https://doi.org/10.1104/pp.105.070326 Crossref, Medline, ISI, Google Scholar
- 2012. Interplay of flg22-induced defence responses and nodulation in Lotus japonicus. J. Exp. Bot. 63:393-401. https://doi.org/10.1093/jxb/err291 Crossref, Medline, ISI, Google Scholar
- 2001. Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula. Plant Cell 13:1835-1849. https://doi.org/10.1105/TPC.010193 Crossref, Medline, ISI, Google Scholar
- 2018. Whole-genome landscape of Medicago truncatula symbiotic genes. Nat. Plants 4:1017-1025. https://doi.org/10.1038/s41477-018-0286-7 Crossref, Medline, ISI, Google Scholar
- 1989. Nodule formation is stimulated by the ethylene inhibitor aminoethoxyvinylglycine. Plant Physiol. 91:690-693. https://doi.org/10.1104/pp.91.2.690 Crossref, Medline, ISI, Google Scholar
- 2019. Expression of the Arabidopsis thaliana immune receptor EFR in Medicago truncatula reduces infection by a root pathogenic bacterium, but not nitrogen-fixing rhizobial symbiosis. Plant Biotechnol. J. 17:569-579. https://doi.org/10.1111/pbi.12999 Crossref, Medline, ISI, Google Scholar
- 2009. Two type III secretion system effectors from Ralstonia solanacearum GMI1000 determine host-range specificity on tobacco. Mol. Plant-Microbe Interact. 22:538-550. https://doi.org/10.1094/MPMI-22-5-0538 Link, ISI, Google Scholar
- 2010. Complete genome sequence of the Medicago microsymbiont Ensifer (Sinorhizobium) medicae strain WSM419. Stand. Genomic Sci. 2:77-86. Crossref, Medline, ISI, Google Scholar,
- 2002. Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415:497-502. https://doi.org/10.1038/415497a Crossref, Medline, ISI, Google Scholar
- 2011. Phosphoproteome analysis of Lotus japonicus roots reveals shared and distinct components of symbiosis and defense. Mol. Plant-Microbe Interact. 24:932-937. https://doi.org/10.1094/MPMI-09-10-0222 Link, ISI, Google Scholar
- 2006. Effects of endogenous salicylic acid on nodulation in the model legumes Lotus japonicus and Medicago truncatula. Plant Physiol. 141:1473-1481. https://doi.org/10.1104/pp.106.080986 Crossref, Medline, ISI, Google Scholar
- 2009. Dissection of bacterial wilt on Medicago truncatula revealed two type III secretion system effectors acting on root infection process and disease development. Plant Physiol. 150:1713-1722. https://doi.org/10.1104/pp.109.141523 Crossref, Medline, ISI, Google Scholar
- 2007. Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume plant Medicago truncatula. Mol. Plant-Microbe Interact. 20:159-167. https://doi.org/10.1094/MPMI-20-2-0159 Link, ISI, Google Scholar
- 2006. Integrated regulation of the type III secretion system and other virulence determinants in Ralstonia solanacearum. PLoS Pathog. 2:e82. https://doi.org/10.1371/journal.ppat.0020082 Crossref, Medline, ISI, Google Scholar
The author(s) declare no conflict of interest.
Funding: B. Gourion was supported by grants ANR-10-LABX-41 and ANR-17-CE20-0013 and the INRAE Département Santé des Plantes et Environnement.