ASSOCIATIONS OF SYMBIOTIC BACTERIA WITH PLANTS
- Albareda Contreras, Marta - Assistant Lecturer
- Alejandre Villalobos, Carla - Student
- Ballesteros Gutiérrez, Marta - PhD Student
- Rey Navarro, Luis - Associate Professor
- Silva de Sousa, Bruna Fernanda - PhD Student
- Vicente Santiago, Noelia - Technician
The association between legume plants and a group of alpha-proteobacteria, collectively known as rhizobia, allows the reduction of atmospheric nitrogen into ammonia in specialized organs called root nodules. This symbiotic nitrogen fixation is carried out by the nitrogenase enzymatic complex synthesized by endosymbiotic Rhizobium cells called bacteroids, which are fed with organic acids derived from plant photosynthate. The Rhizobium-legume symbiosis provides a non-polluting source of nitrogen for legume crops, and constitutes a key component for the development of sustainable agriculture (Power, 2010). Intensive research carried out in many labs during the last decades has revealed that the establishment of the symbiosis involves a sophisticate exchange of signals for specific recognition and metabolic adaptation of both partners (Downie, 2010; van de Velde et al., 2010). It has become clear that the output of the symbiosis is the result of a delicate balance between the two partners, with multiple adaptations involved in both plant and bacteria. The identification of determinants involved in this process will contribute to the elucidation of the fine-tuning of both symbionts to accommodate to each other with potential biotechnological applications in the development of more efficient rhizobial inoculants.
Our lab has been working for a number of years in the study of traits involved in the establishment and functioning of the symbiotic association between rhizobia and legumes, providing new data on the characterization of hydrogenase, a metalloenzyme relevant for the symbiotic process, also studying mechanisms for nickel provision for enzyme biosynthesis and metal homeostasis (Brito et al., 2010; Rubio-Sanz et al., 2013); we have also contributed to the description of novel rhizobia specific for different legumes (Sanchez-Cañizares et al., 2011; Durán et al., 2013). Our current work aims at progressing along some basic questions regarding the Rhizobium-legume symbiosis:
- How do rhizobia adapt to specific conditions within the nodule?
- How does the host plant control bacteria behaviour within the nodule?
- What is the potential of novel Rhizobium-legume symbiotic systems?
We are developing our work along two main research lines:
1) Determinants of the efficiency and specificity of the symbiosis
Our working hypothesis is that differential sets of plant-dependent compounds/conditions control the activity of the bacteria in a host-specific way, and that the bacteria adapts to them by expressing different sets of genes. Also, bacteria modify the behavior of the plant host through effectors sent to the plant via protein secretion systems.
A combination of transcriptomic and proteomic analyses of bacteroids induced in two different legumes (pea and lentil) has been set up to study host-dependent bacterial traits. Previous work at our lab showed that expression of [NiFe] hydrogenase, a metalloenzyme able to recycle the hydrogen evolved by nitrogenase, depends on the identity of the legume host (Brito et al., 2008). In addition to hydrogenase proteins, and based in the availability of the genome of our reference rhizobial strain (Sanchez-Cañizares et al., 2018), we have identified ca. 100 rhizobial proteins differentially expressed on one of the legume hosts. These proteins include several stress-response related (small Heat-Shock Proteins and Universal Stress Proteins), transcriptional regulators, and proteins involved in the C/N metabolism of the bacteroid, among others. These results suggest that the bacteroids are subjected to stress, and also that the identity of the plant host affects the quality of stress within nodules. The effect that other stresses, such as the presence of heavy metals, have in the symbiosis has been also studied (Rubio-Sanz et al., 2018).
A better characterization of host-specific determinants will allow us to define optimal Rhizobium-legume combinations as an approach to obtain elite inoculants. We are currently carrying out functional analysis of some of the identified host-dependent determinants. To do this, mutant derivatives affected on each of the corresponding genes are being analyzed to determine the effect of each trait on symbiotic performance (Duran et al., in preparation). Also, in collaboration with Dr. Fernandez-Pacios, we are pursuing the elucidation of structure-to-function relationships in R. leguminosarum host-dependent hydrogenase-related proteins acting as enzyme subunits, chaperones, scaffolding- and oxygen-protective proteins, and nickel –providing systems (Albareda et al., 2012; 2013; 2014; 2015; Albareda et al., submitted).
On the plant side, we are determining the profile of Nodule-specific peptides (NCR antimicrobial peptides) produced by the plant in the nodule. Over 50 plant-derived NCR peptides were identified in bacteroids isolated from each host. The lack of conserved peptides between both hosts suggest that a strong adaptation has taken place also at the macrosymbiont, indicating the existence of additional levels of plant- dependent control of bacterial behavior.
We are also interested in the role of protein secretion systems in rhizobia. Similarly to many bacterial pathogens, rhizobia are able to inject proteins, called effectors, into the plant cells by means of different protein secretion systems such as type III (T3SS), structurally related to bacterial flagella, and the recently described type VI (T6SS), similar to the tail spike of the T4 phage. We have identified a T3SS-dependent effector (MdcE) that undergoes a Ca-dependent autoproteolysis whose function in the symbiosis remains unknown (Duran et al., 2017). Analysis of specific mutants in T3SS and T6SS present in some of the rhizobia isolated by our research group has revealed a relevant role of secretion systems on the establishment of effective symbiosis and on the definition of host range (Salinero et al., submitted).
2) Biodiversity and symbiotic performance of native microflora nodulating wild legumes in Western Mediterraneum.
Analysis of new symbiotic combinations based on poorly known endemic legumes might provide new applications for the Rhizobium-legume symbiosis. Lupinus (lupines) embraces a very diverse group of legumes including plants with highly protein-rich seeds, which are of great value for human and animal feeding and for improving degraded or nitrogen-poor soils. The study of the symbiosis with these plants is the subject of research carried out in collaboration with the group of Dr. Juan Imperial. Like other legumes, lupines exhibit dinitrogen-fixing capacity through its association with soil rhizobia, resulting in plants with a great value for human and animal feeding and for improving degraded soils or soils with a problematic fertilization.
In contrast to most Lupinus spp., which preferentially thrives in acid soils, Lupinus mariae-josephae grows only in alkaline-lime soils (Sanchez-Cañizares et al., 2011). Bacteria isolated from root nodules of this unique lupine species are being genetically and symbiotically studied, with particular emphasis given to their taxonomy and evolution, and a new bacterial species was recently described (Durán et al., 2013; Ahnia et al., 2018). The role of these bacteria in the survival of the plant under their harsh native conditions has been also studied in collaboration with the Comunidad Valenciana regional government (Navarro et al., 2014).
Also, we are working on the characterization of diazotrophic symbiosis of wild, legume shrubs such as Retama or Cytisus), native in Northern Africa in collaboration with research groups from Algeria and Tunisia. These plants play a key function in the reduction of soil degradation and in the prevention of desert progression. The singular root systems of these plants are ideal for dune stabilization, for the restoration and conservation of ecosystems in arid areas, and in the extension of vegetable cover to desert regions. These legumes associate with a wide diversity of rhizobia that might be useful as inoculants, and we are contributing to their characterization (Msaddak et al, 2017a, 2017b, 2018; Bourebaba et al., 2016). These applied objectives are combined with more basic research on the genomics of these endosymbiotic bacteria, such as the identification of novel effectors potentially secreted through protein secretion systems active under symbiotic conditions.
Salinero-Lanzarote, A., Pacheco-Moreno, A., Domingo-Serrano, L., Durán, D., Ormeño-Orrillo, E., Martínez-Romero, E., Albareda, M., Palacios, J.M., Rey, L. 2019. The type VI secretion system of Rhizobium etli Mim1 has a positive effect in symbiosis. FEMS Microbiology Ecology fiz054. DOI: 10.1093/femsec/fiz054
Albareda, M., Pacios, L.F., Palacios, J.M. 2019. Computational analyses, molecular dynamics, and mutagenesis studies of unprocessed form of [NiFe] hydrogenase reveal the role of disorder for efficient enzyme maturation. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1860, 325–340. DOI: 10.1016/j.bbabio.2019.01.001
Msaddak, A; Rejili, M; Durán, D; Rey, L; Palacios, JM; Imperial, J; Ruiz-Argüeso, T; Mars, M. 2018. "Definition of two new symbiovars, sv. lupini and sv. mediterranense, within the genera Bradyrhizobium and Phyllobacterium efficiently nodulating Lupinus micranthus in Tunisia". Systematic and Applied Microbiology. DOI: 10.1016/j.syapm.2018.04.004".
Ahnia, H; Bourebaba, Y; Durán, D; Boulila, F; Palacios, JM; Rey, L; Ruiz-Argüeso, T; Boulila, A; Imperial, J. 2018. "Bradyrhizobium algeriense sp. nov., a novel species isolated from effective nodules of Retama sphaerocarpa from Northeastern Algeria". Systematic and Applied Microbiology. DOI: 10.1016/j.syapm.2018.03.004".
Sánchez-Cañizares, C; Jorrín, B; Durán, D; Nadendla, S; Albareda, M; Rubio-Sanz, L; Lanza, M; González-Guerrero, M; Prieto, R; Brito, B; Giglio, M; Rey, L; Ruiz-Argüeso, T; Palacios, J; Imperial, J. 2018. "Genomic diversity in the endosymbiotic bacterium Rhizobium leguminosarum". Genes. DOI: 10.3390/genes9020060".
Rubio-Sanz, L; Brito, B; Palacios, J. 2018. "Analysis of metal tolerance in Rhizobium leguminosarum strains isolated from an ultramafic soil". FEMS Microbiology Letters. DOI: 10.1093/femsle/fny010".
Rodrigue, A; Albareda, M; Mandrand-Berthelot, MA; Palacios, J. 2017. "Nickel in Microbial Physiology - From Single Proteins to Complex Trafficking Systems: Nickel Import/Export", p. 237-258. In D. Zamble, M. Rowińska-Żyrek, and H. Kozlowski (eds.), The Biological Chemistry of Nickel. Royal Society of Chemistry. DOI: 10.1039/9781788010580-00237".
Durán, D; Imperial, J; Palacios, J; Ruiz-Argüeso, T; Göttfert, M; Zehner, S; Rey, L. 2017. "Characterization of a novel MIIA domain-containing protein (MdcE) in Bradyrhizobium spp". FEMS Microbiology Letters. DOI: 10.1093/femsle/fnx276".
Msaddak, A; Rejili, M; Duran, D; Rey, L; Imperial, J; Palacios, J; Ruiz-Argueso, T; Mars, M. 2017. "Members of Microvirga and Bradyrhizobium genera are native endosymbiotic bacteria nodulating Lupinus luteus in Northern Tunisian soils". FEMS Microbiology Ecology. DOI: 10.1093/femsec/fix068".
Msaddak, A; Durán, D; Rejili, M; Mars, M; Ruiz-Argüeso, T; Imperial, J; Palacios, J; Rey, L. 2017. "Diverse bacteria affiliated with the genera Microvirga, Phyllobacterium and Bradyrhizobium nodulate Lupinus micranthus growing in soils of Northern Tunisia". Applied and Environmental Microbiology. DOI: 10.1128/aem.02820-16".
Bourebaba, Y; Durán, D; Boulila, F; Ahnia, H; Boulila, A; Temprano, F; Palacios, JM; Imperial, J; Ruiz-Argüeso, T; Rey, L. 2016. "Diversity of Bradyrhizobium strains nodulating Lupinus micranthus on both sides of the Western Mediterranean: Algeria and Spain". Systematic and Applied Microbiology. DOI: 10.1016/j.syapm.2016.04.006".
Baginsky, C; Brito, B; Scherson, R; Pertuzé, R; Seguel, O; Cañete, A; Araneda, C; Johnson, WE. 2015. "Genetic diversity of Rhizobium from nodulating beans grown in a variety of Mediterranean climate soils of Chile". Archives of Microbiology. DOI: 10.1007/s00203-014-1067-y".
Albareda, M; Rodrigue, A; Brito, B; Ruiz-Argueso, T; Imperial, J; Mandrand-Berthelot, M-A; Palacios, J. 2015. "Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake". Metallomics. DOI: 10.1039/C4MT00298A".
Albareda, M.; Pacios, L.F.; Manyani, H.; Rey, L.; Brito, B.; Imperial, J.; Ruiz-Argueso, T.; Palacios, J.M. 2014. "Maturation of Rhizobium leguminosarum hydrogenase in the presence of oxygen requires the interaction of the chaperone HypC and the scaffolding protein HupK". Journal of Biological Chemistry 289, 21217-21229. DOI: 10.1074/jbc.M114.577403".
Sánchez-Cañizares, C; Palacios, J. 2013. "Construction of a marker system for the evaluation of competitiveness for legume nodulation in Rhizobium strains". Journal of Microbiological Methods. DOI: S0167-7012(13)00003-1 [pii] 10.1016/j.mimet.2012.12.022".
Durán, D; Rey, L; Sánchez-Cañizares, C; Navarro, A; Imperial, J; Ruiz-Argüeso, T. 2013. "Genetic diversity of indigenous rhizobial symbionts of the Lupinus mariae-josephae endemism from alkaline-limed soils within its area of distribution in Eastern Spain". Systematic and Applied Microbiology. DOI: S0723-2020(12)00159-2 [pii] 10.1016/j.syapm.2012.10.008".
Ormeño-Orrillo, E; Servín-Garcidueñas, LE; Imperial, J; Rey, L; Ruiz-Argüeso, T; Martínez-Romero, E. 2013. "Phylogenetic evidence of the transfer of nodZ and nolL genes from Bradyrhizobium to other rhizobia". Molecular Phylogenetics and Evolution. DOI: S1055-7903(13)00099-7 [pii] 10.1016/j.ympev.2013.03.003".
Durán, D; Rey, L; Sánchez-Cañizares, C; Jorrín, B; Imperial, J; Ruiz-Argüeso, T. 2013. "Biodiversity of Slow-Growing Rhizobia: The Genus Bradyrhizobium", p. 21-46. In B. Rodelas and J. González (ed.), Beneficial Plant-microbial Interactions: Ecology and Applications. CRC Press.
Rubio-Sanz, L; Prieto, RI; Imperial, J; Palacios, JM; Brito, B. 2013. "Functional and expression analysis of the metal-inducible dmeRF system from Rhizobium leguminosarum bv. viciae". Applied and Environmental Microbiology. DOI: AEM.01954-13 [pii] 10.1128/AEM.01954-13".
Albareda, M.; Manyani, H.; Imperial, J.; Brito, B.; Ruiz-Argueso, T.; Bock, A.; Palacios, J.M. 2012. Dual role of HupF in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum. BMC Microbiology 12:256.