Abstract

Nitrogen-fixing rhizobia bacteria engage in a mutualistic symbiosis with legume plants. One of the defining features of this symbiosis is the formation of organs called nodules on the roots of the hosts. Establishment of an efficient interaction requires sophisticated and bidirectional communication between the host and the microsymbiont. The perception of the rhizobial signals by the host leads to the internalisation of rhizobia on the growing root nodule after the epidermal penetration and cortical spreading steps. The perception of rhizobial signal in the early stages has been extensively explored, however, the internalisation mechanism is still under investigation. This is caused by the lack of genetically amenable systems to study. Plant made tubular like structures, called infection threads are formed during rhizobia infection of host cells. A system that can uncouple infection thread formation and host cell infection will be suitable to uncover the mechanism of the internalisation process. To identify a suitable system to study this mechanism, a natural isolate Rhizobium leguminosarum Norway that infects Lotus was explored. Confocal and electron microscopy uncovered that Rl Norway invades the root nodule of Lotus burttii without using infection threads. Strikingly, Rl Norway is directly internalised from the apoplast into the host cell via “peg”-like structures. The expression of symbiotic genes involved in the infection process induced by Rl Norway is delayed and decreased in comparison to the response induced by a strain utilising an infection thread-dependent mode. These results revealed that Rl Norway uses an alternative infection strategy to colonise Lotus cells. Furthermore, a mutant impaired in the biosynthesis of the Lipochitooligosaccharides, known as Nodulation (Nod) factors, failed to induce “peg”-like structures during the internalisation process. This indicates that the formation of “peg”-like structures depends on the Nod factors and reinforces the previous hypothesis that there is signal perception before rhizobia are internalised. In addition to the signalling exchanges with the host, the rhizobia root colonisation is a prerequisite for the establishment of the root nodule symbiosis. However, the root colonisation of the microbe community is a complex process. The interaction between rhizobia, including competition and cooperation, is hypothesised to influence the root colonisation, which is so far not well examined. Rl Norway was co-isolated with Mesorhizobium norvegicum 10.2.2 from the same nodule. Interestingly, the microscopic quantification of root colonisation revealed increasing colonisation of Rl Norway and Mn 10.2.2 in the co-inoculation compared with the single inoculation. To understand the mechanism underlying the increased root colonisation, rhizobia behaviours related to the root colonisation were determined. A swarming assay showed that the motility of Mn 10.2.2 is increased in the presence of Rl Norway. In addition, biofilms were quantified in vitro. Rl Norway formed biofilms alone, while Mn 10.2.2 did not. Interestingly, co-culture of Rl Norway and Mn 10.2.2 enabled Mn 10.2.2 to form mixed biofilms together with Rl Norway. To investigate the role of the surface polysaccharides of Rl Norway during mixed biofilms formation, the biofilms of a mutant, with impaired surface polysaccharides biosynthesis was analysed. The structure of the biofilms formed by this mutant was altered under the single- and co-inoculation condition in comparison with the wild type strain. This indicates that the structure of the biofilms is determined by the surface polysaccharides of Rl Norway. Overall, this thesis concludes that the two strains exhibit synergism, which could possibly contribute to the increased root colonisation.