Exo-oligosaccharide signaling in Lotus japonicus roots promotes synthesis and secretion of symbiotic phenylpropanoids resulting in increased production of Nod factors in rhizobia.

Besides lipo-chitooligosaccharidic Nod factors (NFs), rhizobial exo-oligosaccharides (EOS), derived from exo-polysaccharides (EPS), are symbiotic signals that promote nodule formation on legume roots. However, plant genes regulated by EOS signaling remain unidentified. Here, we used purified EPS and the rhizobial glycanase ExoK to produce EOS in vitro. EOS was applied to Lotus japonicus roots to identify genome-wide gene expression changes. Mutants of Sinorhizobium sp. NGR234 and L. japonicus were used to investigate the symbiotic roles of EOS and NFs. Transcriptomic analysis showed that EOS induced the expression of many phenylpropanoid biosynthesis genes, among other transcriptome alterations. EOS signaling occurred in both L. japonicus wild-type plants and mutants with impaired NF signaling, but not in epr3 mutant plants defective in the EOS receptor. Root exudates from EOS-treated plants showed increased genistein levels and enhanced activation of nodulation genes in NGR234, leading to higher NF production. Further experiments demonstrated that both EOS and NFs upregulate the expression of genes involved in flavonoid synthesis. Inoculation tests with NGR234 and its mutants revealed that EOS, rather than EPS, plays a role in promoting infection thread development in L. japonicus. In conclusion, we identified L. japonicus genes regulated by EOS signaling and found that EOS can stimulate the production of symbiotic phenylpropanoids that upregulate NF synthesis in rhizobia.