A complex regulatory network governs the expression of symbiotic genes in HH103.

INTRODUCTION

The establishment of the rhizobium-legume nitrogen-fixing symbiosis relies on the interchange of molecular signals between the two symbionts. We have previously studied by RNA-seq the effect of the symbiotic regulators NodD1, SyrM, and TtsI on the expression of the symbiotic genes (the regulon) of HH103 upon treatment with the isoflavone genistein. In this work we have further investigated this regulatory network by incorporating new RNA-seq data of HH103 mutants in two other regulatory genes, and . Both genes code for global regulators with a predominant repressor effect on the regulon, although NodD2 acts as an activator of a small number of HH103 symbiotic genes.

METHODS

By combining RNA-seq data, qPCR experiments, and b-galactosidase assays of HH103 mutants harbouring a gene inserted into a regulatory gene, we have analysed the regulatory relations between the , , , , and genes, confirming previous data and discovering previously unknown relations.

RESULTS AND DISCUSSION

Previously we showed that HH103 mutants in the , , , or genes gain effective nodulation with , a model legume, although with different symbiotic performances. Here we show that the combinations of mutations in these genes led, in most cases, to a decrease in symbiotic effectiveness, although all of them retained the ability to induce the formation of nitrogen-fixing nodules. In fact, the , , and single and double mutants share a set of Nod factors, either overproduced by them or not generated by the wild-type strain, that might be responsible for gaining effective nodulation with .