Abstract
The establishment of a successful symbiotic interaction between rhizobia and their respective legume hosts involves complex molecular interactions between the two prospective partners. The determinants behind the specificity between the legume host and its compatible symbiont are yet to be fully understood. Known determinants of specificity that are produced by the bacterial symbiont include modifications to the chitin backbone of Nod factors, signalling through polysaccharides such as exopolysaccharide (EPS), lipopolysaccharide and cyclic beta glucans, and secreted proteins transported via secretion systems such as the Type I, Type III, and Type IV secretion systems.
The Mesorhizobium loti strain NZP2037 belongs to a group of M. loti strains that exhibit a broad host range that encompasses the legumes Lotus japonicus Gifu and the extended host Lotus pedunculatus. In contrast, the closely related M. loti strain, R7A, belongs to the group of M. loti with a narrow host range that encompasses L. japonicus Gifu but not L. pedunculatus, a host that the strain induces only uninfected nodule primordia on. In addition, truncated EPS in R7A generates an incompatible nodulation phenotype on L. japonicus Gifu, while in NZP2037 the same truncated EPS generates only a delayed nodulation phenotype. These observations suggest that NZP2037 harbours unique genetic determinants and is thus of interest to study on a genetic level. Genetic comparisons between the two closely related strains of M. loti identified genes unique to NZP2037 hypothesised to be involved in its broad host range.
In this study, it was found that genes required for the nodulation of L. pedunculatus and for circumvention of the negative effect of truncated EPS on nodulation of L. japonicus Gifu were encoded on the symbiosis island of NZP2037, as a “hybrid” strain with the chromosome of R7A and the symbiosis island of NZP2037 showed the NZP2037 phenotypes. Genes of interest unique to NZP2037 were targeted for disruption and the effects of these disruptions on nodulation of L. pedunculatus and their relation to the circumvention of the negative effect of defective EPS on nodulation of L. japonicus were investigated. The genes nodU, nodFEG, and nodA2 were expendable for nodulation and were not involved in the circumvention of defective EPS signalling. Interestingly, nodU was found to promote nodule organogenesis, but by itself was not enough to restore nodulation to R7A EPS mutants. Similarly, additional genetic targets unique to NZP2037 relating to effectors of the Type IV secretion system and a Nod factor gene mln399 were also disrupted. These genes were also expendable for nodulation and were not involved in the circumvention of defective EPS signalling. Furthermore, a strain with mutations in all of the above genes showed the same phenotype as NZP2037, eliminating the possibility that the genes were functionally redundant for nodulation.
The major finding in this study was the involvement of the Type I secretion system PrsDE and its respective effector Mln031 in the nodulation of the extended host L. pedunculatus. Mutants defective in prsDE had the unusual phenotype of inducing effective nodules on approximately half of the L. pedunculatus plants inoculated, and only uninfected nodule primordia on the other half. In addition, the initial gene of interest nodO was found to not be involved in the nodulation of the hosts tested. However, the presence of a construct encoding prsDE and mln031 was unable to extend the host range of R7A to include that of L. pedunculatus, suggesting that other molecular signals must be required for the establishment of a successful symbiotic relationship with this host.
The results from this project provides insights into the unique Nod factor modifications of NZP2037 and how they and the Type IV secretion system are expendable for nodulation of the extended host L. pedunculatus and circumvention of defective EPS signalling. The importance of the Type I secretion system PrsDE and Mln031 are highlighted as important determinants of symbiosis bringing the importance of these systems into the forefront of rhizobium-legume interactions.