RIVET-a Tool for in Vivo Analysis of Symbiotically Relevant Gene Expression in Sinorhizobium Meliloti

Despite significant advances in the development of sensitive tools for studying genetics and signal exchange in legume-rhizobium symbioses, many uncertainties remain about the in vivo role of bacterial and plant signals in symbiotic gene regulation. In this study, we adapted TnpR recombinase-based in vivo expression technology (RIVET) to document gene regulation in Sinorhizobium meliloti. The substrate for TnpR, the res1-tet-res1 cassette, is stably inherited when cloned into a neutral site of the S. meliloti genome. Bicistronic promoterless tnpR-beta-glucuronidase (GUS) reporters were constructed to track expression ("resolution") of symbiotically relevant S. meliloti genes during different stages of the interaction. In proof of principle experiments, the resolution of the nodC::tnpR reporter was detected within 4 h of exposure to micromolar levels of the nod operon inducer luteolin and after overnight incubation in the rhizosphere. RIVET demonstrated that cell division gene ftsZ2 was not strongly expressed in the rhizosphere but was activated inside the nodules and on agar surfaces. Rhizosphere expression of the N-acyl homoserine lactone (AHL) synthase sinI::tnpR-GUS reporter was modest in prequorate microcolonies, and then increased with time. AHL synthase sinI and an AHL-regulated gene, expG, were activated inside the nodules.