|dc.description.abstract||The establishment of a successful symbiotic relationship between legumes and rhizobia requires complex molecular communication between the two partners. Rhizobial exopolysaccharide (EPS) has been implicated as a signalling molecule in this process. Wild-type Mesorhizobium loti strain R7A synthesises and secretes an O-actylated acidic EPS composed of octameric subunits comprising a backbone of one galactose and three glucose (Glc) residues, with a branch of two Glc, one glucuronic acid (GlcA) and a terminal riburonic acid (RibA). In a previous study, it was proposed that wild-type R7A EPS plays a signalling role promoting infection thread (IT) formation and the development of nitrogen-fixing nodules. Mutants that were absent of EPS also allowed nodule development. In contrast, truncated versions of R7A EPS produced by mutants affected in the synthesis of the EPS branch was proposed to impair IT development by inducing plant defence responses. The main aim of this study was to further test this hypothesis. In addition, previously unknown genes responsible for the addition of GlcA and RibA were identified in this study.
Further evidence for the two classes of EPS biosynthesis mutants was provided by the characterisation of symbiotically-proficient suppressor mutants of an EPS branch mutant (R7AΔexoU). The suppressor mutants were previously isolated via a transposon mutagenesis screen. It was found that the suppressor mutations were not caused by the transposon insertion but instead were second-site mutations in genes involved in the EPS backbone synthesis, including exoB, exoA, exoL and exoYF. Suppressor mutants isolated in a second transposon mutagenesis screen of R7AΔexoU provided a similar result. Overall, suppressor strains with mutations in EPS backbone synthesis were isolated whereas no EPS branch or transport mutants were found. These results support the hypothesis that R7AΔexoU is symbiotically impaired because it secretes a truncated EPS.
Isolation and characterisation of further R7A EPS biosynthesis mutants provided additional evidence for two phenotypic classes of EPS synthesis mutants. A delay in the formation of nitrogen-fixing nodules was observed on Lotus plants inoculated with R7A mutants affected in synthesis of EPS backbone compared to plants inoculated with R7A wild-type, in contrast to previous results. Closer examination of symbiotic ability of an EPS backbone mutant (R7AΔexoYF) revealed a reduction in IT numbers to half the number formed by R7A wild-type at 14 days post infection and delayed infection of already formed nodule primordia.
The isolation of a mutation in a gene responsible for EPS transport (exoT) within R7AΔexoYF and the failure to isolate such a mutation in the R7A or R7AexoU backgrounds provided evidence that R7AΔexoYF does not secrete any EPS and suggested that failure to secrete wild-type or truncated EPS out of the cell was lethal. A model for EPS transport in R7A was developed.
Two genes, mlr5268 and mll5269, were identified as candidate genes for the addition of GlcA and RibA to R7A EPS. Isolation of mutations in these novel genes and the structural characterisation of EPS synthesised by the mutants confirmed that mlr5268 and mll5269 were responsible for addition of GlcA and RibA, respectively. R7A5269 produced high molecular weight EPS and was symbiotically proficient, suggesting the RibA component of R7A EPS is not required for polymerisation of the octameric subunit, nor the signalling role of EPS. The symbiotic phenotype of R7A5268 was that expected for a EPS branch mutant secreting a truncated EPS.
Taken together, the results in this study support a signalling role for M. loti EPS, suggesting that wild-type EPS provides an enhanced positive signal promoting a more efficient infection process of Lotus plants. Mutants that affect the synthesis of the EPS backbone portion and therefore do not secrete any EPS, are able to form nitrogen-fixing nodules but are not as efficient because they lack the EPS signal. In contrast, mutants involved in the synthesis of the backbone portion (excluding the RibA residue) fail to form nitrogen-fixing nodules, due to the secretion of a truncated version of EPS which provides a negative signal.||