Modulation of Quorum Sensing as an Adaptation to Nodule Cell Infection during Experimental Evolution of Legume Symbionts

Abstract

Over millions of years, changes have occurred in regulatory circuitries in response to genome reorganization and/or persistent changes in environmental conditions. How bacteria optimize regulatory circuitries is crucial to understand bacterial adaptation. Here, we analyzed the experimental evolution of the plant pathogen Ralstonia solanacearum into legume symbionts after the transfer of a natural plasmid encoding the essential mutualistic genes. We showed that the Phc quorum sensing system required for the virulence of the ancestral bacterium was reconfigured to improve intracellular infection of root nodules induced by evolved Ralstonia. A single mutation in either the PhcB autoinducer synthase or the PhcQ regulator of the sensory cascade tuned the kinetics of activation of the central regulator PhcA in response to cell density so that the minimal stimulatory concentration of autoinducers needed for a given response was increased. Yet, a change in the expression of a PhcA target gene was observed in infection threads progressing in root hairs, suggesting early programming for the late accommodation of bacteria in nodule cells. Moreover, this delayed switch to the quorum sensing mode decreased the pathogenicity of the ancestral strain, illustrating the functional plasticity of regulatory systems and showing how a small modulation in signal response can produce drastic changes in bacterial lifestyle. IMPORTANCE Rhizobia are soil bacteria from unrelated genera able to form a mutualistic relationship with legumes. Bacteria induce the formation of root nodules, invade nodule cells, and fix nitrogen to the benefit of the plant. Rhizobial lineages emerged from the horizontal transfer of essential symbiotic genes followed by genome remodeling to activate and/or optimize the acquired symbiotic potential. This evolutionary scenario was replayed in a laboratory evolution experiment in which the plant pathogen Ralstonia solanacearum successively evolved the capacities to nodulate Mimosa pudica and poorly invade, then massively invade, nodule cells. In some lines, the improvement of intracellular infection was achieved by mutations modulating a quorum sensing regulatory system of the ancestral strain. This modulation that affects the activity of a central regulator during the earliest stages of symbiosis has a huge impact on late stages of symbiosis. This work showed that regulatory rewiring is the main driver of this pathogeny-symbiosis transition.

Experimental Evolution of Legume Symbionts: What Have We Learnt?

“…Secondly, we observed a coupling between infection and nodulation since all mutations that improved intracellular infection also improved nodulation competitiveness [22] (and unpublished data). The genetic link between infection and nodulation is supported by the finding that mutations optimizing infection in our EE modify the expression of bacterial genes during the earliest stages of symbiosis (infected root hairs) [24]. This coupling is also consistent with the known effect of Nod factors on intracellular infection [126][127][128] and the fact that the intracellular release of rhizobia in primodium cells depends on the proper progression of infection threads [129], which otherwise conditions nodule development.…”

“…This second level of infection was obtained via mutations affecting global transcription regulators, either EfpR or PhcA. Missense mutations in efpR itself or an intergenic mutation upstream from a gene of unknown function triggered a constitutive repression of EfpR [23], while mutations in two different components of the Phc quorum sensing system (phcB and phcQ) led to a modulation of the quorum sensing threshold activating PhcA [24]. EfpR and PhcA were shown to act as both central players of the R. solanacearum virulence regulatory network and global catabolic repressors [23,[87][88][89].…”

“…a future challenge will be to identify which functions controlled by the targeted regulators are beneficial or deleterious for intracellular infection in the Ralstonia-Mimosa interaction. We hypothesize that the same functions are affected in the different lines, but the large overlap between the EfpR and PhcA regulons [24] makes this task difficult (Figure 3). .…”

“…a first level of intracellular infection was obtained by the inactivation of the virulence regulator HrpG [21,22]. Optimized intracellular infection was reached either by inactivation of the EfpR pathway or a modulation of the quorum sensing pathway controlling PhcA [23,24]. EfpR and PhcA controls a common set of genes as well as other specific genes.…”

“…Three levels of infection were observed in the evolved lines: extracellular, partially intracellular and nicely intracellular. The main adaptive mutations responsible for the acquisition and improvement of symbiosis were identified [21][22][23][24]. *, stop mutation.…”