Abstract
Rhizobia are a group of soil bacteria that form nitrogen-fixing symbioses with legumes through the formation of root nodules. The Mesorhizobium-Lotus symbiosis is one model used to study how these relationships are established. Mesorhizobium spp. acquire genes necessary for symbiosis through horizontal transfer of integrative and conjugative elements (ICEs) or symbiosis islands. Differences in the genes carried on these ICEs contribute to differences in the host-range and effectiveness of the symbiosis with Lotus. Mesorhizobium spp. strains can also be classified into two host-range groups based on their ability to nodulate Lotus pedunculatus. Group I strains (e.g. M. japonicum R7A) only induce uninfected nodule primordia on this host, while Group II strains (e.g. M. jarvisii NZP2037) efficiently induce effective nodules. Both groups can effectively nodulate Lotus japonicus Gifu.
The symbiosis is established through a complex exchange of signalling molecules between the host and symbiont. Among these are exopolysaccharides (EPSs), which are structurally diverse polymers secreted by rhizobia and recognised by the plant to promote an effective symbiosis. In L. japonicus Gifu, the EPR3 and EPR3a EPS receptors perceive rhizobial EPS to modulate the symbiosis. The receptors are thought to respond positively to correct forms of EPS to enhance symbiosis and respond negatively to incorrect forms to inhibit symbiosis. R7A∆exoU is an EPS mutant that produces a truncated form of EPS which is negatively perceived by the receptors and results in formation of uninfected nodule primordia. However, a TC2037exoU mutant is able to form effective nodules. TC2037 is a transconjugant strain that harbours the NZP2037 symbiosis island and core chromosome of R7A. Both R7A and TC2037 exoU mutants are expected to produce the same truncated EPS, as genes required for EPS biosynthesis are chromosomally located. This suggested that a gene(s) involved in EPS regulation is present on the NZP2037 island, and absent from the R7A island, which may negate the negative signalling of truncated EPS.
Comparative analyses revealed several genes present on the GII islands that were absent from the GI symbiosis. Of particular interest was syrA, which encodes a small cytoplasmic membrane protein involved in EPS regulation. Both GI and GII islands contained a copy of syrA (syrA1) located downstream of nitrogen fixation genes. However, only GII islands contained a second copy of syrA (syrA2) that has been translocated downstream of nodulation genes, indicating it is likely expressed during early symbiosis in response to plant flavonoids. In Sinorhizobium meliloti, SyrA increases EPS production during both free-living and symbiotic states. In contrast, this study showed that Mesorhizobium spp. SyrA represses EPS production during the symbiotic state. The expression of syrA2 during early symbiosis reduced the nodulation efficiency of NZP2037 on L. japonicus Gifu but not L. pedunculatus, suggesting syrA2 may act as a broad-host-range determinant of Mesorhizobium GII strains. Additionally, evidence was gained that another signalling component of L. japonicus Gifu may also respond to EPS in addition to EPR3 and EPR3a, and that EPR3a may negatively regulate this component during symbiosis.
This study also investigated the interacting partner of Mesorhizobium spp. SyrA in order to understand how two closely-related proteins have evolved opposite effects on EPS production. I revealed that Mesorhizobium spp. SyrA does not interact with the ExoR-ExoS-ChvI system as proposed for S. meliloti SyrA and instead may interact with a phosphoglycosyl transferase, ExoY, analogous to the interaction confirmed herein for another EPS repressor, ExoX. Using Alpha-Fold protein-protein interaction models, bacterial two-hybrids and amino-acid mutagenesis, evidence was obtained that Mesorhizobium spp. SyrA and ExoX may prevent ExoY from recognising or transferring the first phosphosugar to a membrane-bound polyprenol phosphate required for the initiation of the EPS biosynthetic pathway.
Further analysis also revealed that Mesorhizobium spp. harbour multiple homologues of ExoX, which interact with different ExoY homologues involved in the initiation of various polysaccharide biosynthetic pathways, implying the presence of a complex regulatory network that remains to be explored.