Projects

Funding: SNF project 31003A_179322 (2018-2023)
People involved: Dr. Paula Bellés Sancho, Dr. Yilei Liu, Romy Leemann
Collaborators: Dr. Christian Ahrens (Agroscope-Molecular Ecology and Swiss Institute of Bioinformatics), Prof. Nicola Zamboni (IMSB, ETHZ).

The successful interaction between specific rhizobia and host plants lead to the formation of specialized symbiotic organs, called root nodules, in which the bacteria fix atmospheric nitrogen (N₂) and make it available to the plant. In exchange, the plant provides carbon and energy sources generated by photosynthesis. A complex series of events, coordinated by both plant and bacterial signals, underlies the development of this symbiotic interaction¹. Until recently, rhizobia were thought to belong exclusively to the alpha-subclass of proteobacteria (alpha-rhizobia, isolated first in 1888). This changed in 2001, when two genera belonging to the beta-proteobacteria (Burkholderia and Cupriavidus) were discovered (beta-rhizobia)². For the legume nodulating strains belonging to the environmental clade of the genus Burkholderia, recently a new genus, Paraburkholderia, has been proposed^. The strain Paraburkholderia phymatum STM815 stands out for its ability to establish a nitrogen-fixing symbiosis with more than 50 different legumes (including mimosoid and papilionoid) as well as to reduce nitrogen in free-living conditions (Figure 1a,b)^3–8. Moreover, the availability of the complete genome sequence makes it an ideal model to characterize the whole gene/protein expression profile during symbiosis⁵.

In order to understand the molecular mechanism underlying beta-rhizobia—legume symbiosis and identify differences and commonalities between alpha- and beta-rhizobia, we use functional genomic approaches such as transposon sequencing (Tn-Seq), RNA-sequencing, shotgun proteomics and metabolomics (Figure 1c). Our group was the first to determine the whole transcriptome profile of P. phymatum STM815 under nitrogen limitation (a condition that partially mimics the condition encountered by free-living rhizobia in nitrogen-starved soils) and in symbiosis with the economically important papilionoid legume Phaseolus vulgaris (common bean)⁶. Recently, the combination of metabolomics and dual RNA-sequencing approach on nodules allowed us to identify novel gene functions such as the repression of the production of indole-3-acetic acid (IAA) by the master activator of nitrogen-fixation NifA⁹.

The goal of this project is to identify and characterize beta-rhizobial genes important in symbiotic partner recognition, symbiosis establishment, and N₂-fixation.

Figure 1.a) Common bean root-nodules induced and colonized by P. phymatum STM815. b) Section of common bean nodules colonized by P. phymatum, plant cell walls stained with calcofluor white (green) and bacterial cells with Syto9 DNA (magenta) (picture from Bellés-Sancho et al., 2022 (Frontiers in Plant Science)). c) Functional genomic approaches used with P. phymatum.

References:

1. Bellés-Sancho, P., Beukes, C., James, E. K. & Pessi, G. Nitrogen-fixing symbiotic Paraburkholderia species: current knowledge and future perspectives. Nitrogen 4, 135–158 (2023).
2. Moulin, L., Munive, A., Dreyfus, B. & Boivin-Masson, C. Nodulation of legumes by members of the beta-subclass of Proteobacteria. Nature 411, 948–950 (2001).
3. Elliott, G. N. et al. Burkholderia phymatum is a highly effective nitrogen-fixing symbiont of Mimosa spp. and fixes nitrogen ex planta. New Phytologist 173, 168–180 (2007).
4. Talbi, C. et al. Burkholderia phymatum strains capable of nodulating Phaseolus vulgaris are present in moroccan soils. Appl Environ Microbiol 76, 4587–4591 (2010).
5. Moulin, L. et al. Complete Genome sequence of Burkholderia phymatum STM815^T, a broad host range and efficient nitrogen-fixing symbiont of Mimosa species. Stand Genomic Sci 9, 763–774 (2014).
6. Lardi, M., Liu, Y., Purtschert, G., de Campos, S. B. & Pessi, G. Transcriptome analysis of Paraburkholderia phymatum under nitrogen starvation and during symbiosis with Phaseolus vulgaris. Genes (Basel) 8, (2017).
7. Lardi, M., de Campos, S. B., Purtschert, G., Eberl, L. & Pessi, G. Competition experiments for legume infection identify Burkholderia phymatum as a highly competitive β-rhizobium. Front Microbiol 8, (2017).
8. Bellés-Sancho, P. et al. Paraburkholderia phymatum homocitrate synthase NifV plays a key role for nitrogenase activity during symbiosis with papilionoids and in free-living growth conditions. Cells 10, 952 (2021).
9. Bellés-Sancho, P. et al. A novel function of the key nitrogen-fixation activator NifA in beta-rhizobia: Repression of bacterial auxin synthesis during symbiosis. Front Plant Sci 13, (2022).

Funding: SNF project 31003A_179322 (2018-2023)
People involved: Dr. Paula Bellés Sancho, Sebastian Hug, Kim Bolli, Yilei Liu

Rhizobia must endure the harsh conditions encountered in the soil to be able to colonize plant roots. Drought, pH, salinity, temperature, and the presence of toxic substances are some examples of environmental stresses that rhizobia must withstand, as are biotic factors such as the presence of other bacteria species competing for common resources. Therefore, the competitiveness of a rhizobia strain is directly influencing a successful colonization of the plant¹.

STM815 stands out among the beta-rhizobia group for being the most competitive strain in vitro and for nodulating papilionoid legumes^2–4. Our primary goal is to elucidate the strategies and identify traits that P. phymatum employs to outcompete other strains. In our group we showed that P. phymatum has two types 6 secretion systems (T6SS), one of which contributes to outcompete other beta-rhizobia strains in a contact-dependent manner^5,6. Moreover, P. phymatum produced more exopolysaccharides (EPS) and was more motile compared to other strains, two traits crucial for competitiveness (Figure 1)⁴. We also propose that the overproduction of the hormone auxin by this strain may enhance the occurrence of infection events and, as result, improve the frequency of root colonization⁴. Moreover, we recently show that another beta-rhizobial strain, Paraburkholderia sabiae, shows T6SS-dependent antagonistic activity towards important plant pathogens such as Pseudomonas syringae and Pectobacterium carotovorum. Moreover, co-inoculation of potato tubers with P. sabiae resulted in a drastic reduction of soft rot caused by P. carotovorum and P. sabiae’s protective effect was partly dependent on T6SS-1⁷

Figure 1. Phenotypic traits of P. phymatum and P. sabiae that provide a competitive advantage against other bacteria and for root nodulation.

The aim of this project consists in investigating the molecular mechanisms underlying the successful competitiveness of P. phymatum and P. sabiae.

References:

1. Bellés-Sancho, P., Beukes, C., James, E. K. & Pessi, G. Nitrogen-fixing symbiotic Paraburkholderia species: current knowledge and future perspectives. Nitrogen 4, 135–158 (2023).
2. Elliott, G. N. et al. Burkholderia spp. are the most competitive symbionts of Mimosa, particularly under N-limited conditions. Environ Microbiol 11, 762–778 (2009).
3. Moulin, L. et al. Complete Genome sequence of Burkholderia phymatum STM815^T, a broad host range and efficient nitrogen-fixing symbiont of Mimosa species. Stand Genomic Sci 9, 763–774 (2014).
4. Lardi, M., de Campos, S. B., Purtschert, G., Eberl, L. & Pessi, G. Competition experiments for legume infection identify Burkholderia phymatum as a highly competitive β-rhizobium. Front Microbiol 8, (2017).
5. de Campos, S. B., Lardi, M., Gandolfi, A., Eberl, L. & Pessi, G. Mutations in two Paraburkholderia phymatum type VI secretion systems cause reduced fitness in interbacterial competition. Front Microbiol 8, 2473 (2017).
6. Hug, S. et al. Differential expression of Paraburkholderia phymatum type VI secretion systems (T6SS) suggests a role of T6SS-b in early symbiotic interaction. Front Plant Sci 12, (2021).
7. Hug, S. et al. Paraburkholderia sabiae uses one type VI secretion system (T6SS-1) as a powerful weapon against notorious plant pathogens. Microbiol Spectr Jul, e01622-23 (2023)

Funding: SNF project 31003A_179322 (2018-2023)
People involved: Dr. Paula Bellés Sancho, Sebastian Hug, Kim Bolli, Yilei Liu

Rhizobia must endure the harsh conditions encountered in the soil to be able to colonize plant roots. Drought, pH, salinity, temperature, and the presence of toxic substances are some examples of environmental stresses that rhizobia must withstand, as are biotic factors such as the presence of other bacteria species competing for common resources. Therefore, the competitiveness of a rhizobia strain is directly influencing a successful colonization of the plant¹.

P. phymatum STM815 stands out among the beta-rhizobia group for being the most competitive strain in vitro and for nodulating papilionoid legumes^2–4. Our primary goal is to elucidate the strategies and identify traits that P. phymatum employs to outcompete other strains. In our group we showed that P. phymatum has two types 6 secretion systems (T6SS), one of which contributes to outcompete other beta-rhizobia strains in a contact-dependent manner^5,6. Moreover, P. phymatum produced more exopolysaccharides (EPS) and was more motile compared to other strains, two traits crucial for competitiveness (Figure 1)⁴. We also propose that the overproduction of the hormone auxin by this strain may enhance the occurrence of infection events and, as result, improve the frequency of root colonization⁴. Moreover, we recently show that another beta-rhizobial strain, Paraburkholderia sabiae, shows T6SS-dependent antagonistic activity towards important plant pathogens such as Pseudomonas syringae and Pectobacterium carotovorum. Moreover, co-inoculation of potato tubers with P. sabiae resulted in a drastic reduction of soft rot caused by P. carotovorum and P. sabiae’s protective effect was partly dependent on T6SS-1⁷

Figure 1. Important PGPR traits present in P. phymatum STM815.
We will use a highly-saturated library of transposon mutants (Tn-seq) and gene expression studies (RNA-seq) to identify and characterize genes involved in P. phymatum response to climate change stresses (high temperatures and soil acidity) as well as coping with common bean root exudates produced under the same stress conditions.

References:

1. Iriti, M. & Vitalini, S. Plant metabolomics in the global scenario of food security: A systems-biology approach for sustainable crop production. Int J Mol Sci 19, (2018).
2. Arora, N. K., Verma, M. & Mishra, J. Rhizobial Bioformulations: Past, Present and Future. in Rhizotrophs: Plant Growth Promotion to Bioremediation (ed. Mehnaz, S.) 69–99 (Springer Singapore, 2017). doi:10.1007/978-981-10-4862-3_4.
3. Moulin, L. et al. Complete Genome sequence of Burkholderia phymatum STM815^T, a broad host range and efficient nitrogen-fixing symbiont of Mimosa species. Stand Genomic Sci 9, 763–774 (2014).
4. Lardi, M., de Campos, S. B., Purtschert, G., Eberl, L. & Pessi, G. Competition experiments for legume infection identify Burkholderia phymatum as a highly competitive β-rhizobium. Front Microbiol 8, (2017).
5. Elliott, G. N. et al. Burkholderia spp. are the most competitive symbionts of Mimosa, particularly under N-limited conditions. Environ Microbiol 11, 762–778 (2009).
6. de Campos, S. B., Lardi, M., Gandolfi, A., Eberl, L. & Pessi, G. Mutations in two Paraburkholderia phymatum type VI secretion systems cause reduced fitness in interbacterial competition. Front Microbiol 8, 2473 (2017).
7. dos Reis Junior, F. B. et al. Nodulation and nitrogen fixation by Mimosa spp. in the Cerrado and Caatinga biomes of Brazil. New Phytologist 186, 934–946 (2010).
8. Talbi, C. et al. Burkholderia phymatum improves salt tolerance of symbiotic nitrogen fixation in Phaseolus vulgaris. Plant Soil 367, 673–685 (2013).
9. Melkonian, R. et al. The geographical patterns of symbiont diversity in the invasive legume Mimosa pudica can be explained by the competitiveness of its symbionts and by the host genotype. Environ Microbiol 16, 2099–2111 (2014).
10. Lardi, M., Liu, Y., Purtschert, G., de Campos, S. B. & Pessi, G. Transcriptome analysis of Paraburkholderia phymatum under nitrogen starvation and during symbiosis with Phaseolus vulgaris. Genes (Basel) 8, (2017).
11. Hug, S. et al. Differential expression of Paraburkholderia phymatum type VI secretion systems (T6SS) suggests a role of T6SS-b in early symbiotic interaction. Front Plant Sci 12, (2021).
12. Bellés-Sancho, P. et al. Metabolomics and dual RNAsequencing on root nodules revealed new cellular functions controlled by Paraburkholderia phymatum NifA. Metabolites 11, 455 (2021).
13. Bellés-Sancho, P. et al. A novel function of the key nitrogen-fixation activator NifA in beta-rhizobia: Repression of bacterial auxin synthesis during symbiosis. Front Plant Sci 13, (2022).
14. Elliott, G. N. et al. Burkholderia phymatum is a highly effective nitrogen-fixing symbiont of Mimosa spp. and fixes nitrogen ex planta. New Phytologist 173, 168–180 (2007).
15. Bellés-Sancho, P. et al. Paraburkholderia phymatum homocitrate synthase NifV plays a key role for nitrogenase activity during symbiosis with papilionoids and in free-living growth conditions. Cells 10, 952 (2021).
16. Klonowska, A., Melkonian, R., Miché, L., Tisseyre, P. & Moulin, L. Transcriptomic profiling of Burkholderia phymatum STM815, Cupriavidus taiwanensis LMG19424 and Rhizobium mesoamericanum STM3625 in response to Mimosa pudica root exudates illuminates the molecular basis of their nodulation competitiveness and symbiotic evolutionary history. BMC Genomics 19, (2018)

Funding: Josef Huwyler Ruth Bernet-Engeli Foundation and ESA (I-2022-03207) co-funded Research Idea (2022-2024)
People involved: Daphné Golaz
Collaborators: Prof. Dr. Marcel Egli (IMT, HSLU), Dr. Christian Ahrens (Agroscope-Molecular Ecology and Swiss Institute of Bioinformatics), Prof. Nicola Zamboni (IMSB, ETHZ).

The exploitation of the mutualistic relationship between rhizobia and legumes is a solution to ensure a constant supply in organic nitrogen and an acceptable yield in plants grown in prospective extraterrestrial colonies. To understand the effect of exposure to space-like conditions such as reduced gravity in free-living rhizobia, we are combining classical phenotypic analyses with functional genomics approaches in the beta-rhizobia P. phymatum grown in an artificial microgravity environment. The integration of data gathered through multiples next-generation sequencing methods, namely Tn-Seq, RNA-Seq, shotgun proteomic and metabolomic analyses, allows us to identify and prioritize genes and metabolomic pathways that are key for rhizobia thriving in simulated microgravity. Mutants for the genes of interest are constructed and phenotypically characterized in vitro - for growth, cell morphology, resistance to abiotic stresses and secondary metabolites production - and in planta with the model legume Mimosa pudica for assessment of their symbiotic abilities.