β-rhizobial legume symbiosis
Funding: SNF project 31003A_153374 (2014-2018)
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 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 interaction.
Recently, it was discovered that in addition to the well-studied and phylogenetically diverse α-subclass of proteobacteria (α-rhizobia), certain β-proteobacteria such as Burkholderia and Cupriavidus species (β-rhizobia), are also capable of establishing nitrogen-fixing symbiosis with legumes. Phylogenetic analyses based on key symbiosis genes such as nif (nitrogen fixation) and nod (nodulation) indicate that β-rhizobia have existed as legume symbionts for approximately 50 million years and suggest that they have evolved separately from the well-studied α- rhizobia.
Our research aims to answer the following fundamental questions:
- What are the molecular determinants that are required to successfully establish a symbiosis between legumes and β-rhizobia?
- Are the mechanisms different compared to those used by α-rhizobia?
- Are β-rhizobia able to compete with α- rhizobia for root nodule occupancy?
Functional genomics to unravel the molecular basis of the nitrogen-fixing symbiosis between legumes and β-rhizobia
To understand the molecular processes underlying β-rhizobial symbiosis we are applying a multi-layered functional genomics analysis on root nodules and free-living rhizobia by comparative genomics, RNA-Seq, shotgun proteomics and metabolomics (Figure 1). These state-of-the-art technologies combined with classical phenotypical approaches allow us to identify 1) β-rhizobial genes/proteins important for symbiosis and 2) mechanistic differences between α- and ß-rhizobial symbiosis. After 50 million years of separate evolution we expect that α- vs. β-rhizobial symbiosis should be very different. We use the symbiosis between B. phymatum STM815 and the legume Phaseolus vulgaris (bean) as a model.
Integration of transcriptomics, proteomics and metabolomics datasets derived from the global studies followed by a careful bioinformatic analysis through classification of genes in functional categories, assignment to pathways, gene-set enrichment analysis (GSEA) and literature searches will be used to prioritize Burkholderia genes differentially or even specifically expressed during symbiosis and potentially important for symbiosis with legumes. Mutants in genes of interest will be generated and characterized in vitro for growth, production of secondary metabolites, resistance to environmental stress and in vivo for the capability to efficiently enter symbiosis with legumes (Figure 2).
Competition of α- and β-rhizobia for infection
People involved: Martina Lardi
Collaborators: Prof. Hans-Martin Fischer (ETHZ), Dr. Lionel Moulin/Dr. Agnieszka Klonowska (IRD, Monpellier)
Several legume plants can be efficiently nodulated by α- and β-rhizobia. To answer the question whether α- and β-rhizobia are able to compete for nodule occupancy, we are performing co-inoculation experiments using different α- and β-rhizobial strains and different legumes under different environmental conditions. To this end, differentially tagged strains are used in competition assays. This will allow to follow the interaction and to determine the distribution and proportion of α- and β- rhizobial strains inside the root nodules using a confocal laser microscope (Leica TCS SPE). With these experiments, we aim to identify the genetic determinants relevant for competitive nodulation.
Functional genomics of Burkholderia species
To advance our knowledge of the regulatory mechanisms underlying symbiosis (s. above) or pathogenicity (in the case of opportunistic pathogens) in different Burkholderia strains, a systems biology approach has been established in our laboratory. The recently developed RNA-Seq deep sequencing technology is used to map and quantify the bacterial transcriptome in a given strain or condition (e.g. micro-oxia and low nutrients). In parallel, protein extracts from matched samples are being analyzed with a shotgun proteomics approach to identify the expressed proteome, with a particular focus on achieving a high coverage of the membrane proteome. The quantitative data obtained from RNA-Seq and the semi-quantitative spectral count data from the proteomics approach are integrated. It is assumed that genes that are differentially up-regulated in a micro-oxic and nutrient limited environment mimicking the in vivo situation are important for life and success of Burkholderia strains in the natural environment. To verify a role of the identified genes we generate respective knock-out mutants by the aid of a gene replacement procedure and test the mutants for their symbiotic properties or for virulence using different in vivo infection models. This system biology approach will allow us to better understand the molecular basis of successful Burkholderia – host interactions allowing us to prioritize competitive traits and potential drug targets.