Abstract
Rhizobia, the nitrogen-fixing symbionts of legumes, are polyphyletic bacteria distributed in many alpha- and beta-proteobacterial genera. They likely emerged and diversified through independent horizontal transfers of key symbiotic genes. To replay the evolution of a new rhizobium genus under laboratory conditions, the symbiotic plasmid of Cupriavidus taiwanensis was introduced in the plant pathogen Ralstonia solanacearum, and the generated proto-rhizobium was submitted to repeated inoculations to the C. taiwanensis host, Mimosa pudica L. This experiment validated a two-step evolutionary scenario of key symbiotic gene acquisition followed by genome remodeling under plant selection. Nodulation and nodule cell infection were obtained and optimized mainly via the rewiring of regulatory circuits of the recipient bacterium. Symbiotic adaptation was shown to be accelerated by the activity of a mutagenesis cassette conserved in most rhizobia. Investigating mutated genes led us to identify new components of R. solanacearum virulence and C. taiwanensis symbiosis. Nitrogen fixation was not acquired in our short experiment. However, we showed that post-infection sanctions allowed the increase in frequency of nitrogen-fixing variants among a non-fixing population in the M. pudica–C. taiwanensis system and likely allowed the spread of this trait in natura. Experimental evolution thus provided new insights into rhizobium biology and evolution.
Highlights
The diversity of current living forms is the result of evolutionary processes that have unfolded over billions of years but are difficult to trace in the absence of fossils
In addition to the nod and nif-fix genes involved in Nod Factor synthesis and nitrogen fixation respectively, the pRalta plasmid carries a mutagenesis cassette that elevates mutation rate when bacteria are free-living in the plant culture medium
experimental evolution (EE) did not address the question of the origin of the first rhizobia, nor that of the co-evolution between plants and rhizobia
Summary
The diversity of current living forms is the result of evolutionary processes that have unfolded over billions of years but are difficult to trace in the absence of fossils. The introduction of the symbiotic plasmid of C. taiwanensis LMG19424 into the plant pathogenic Ralstonia solanacearum GMI1000 strain generated a still pathogenic strain unable to nodulate Mimosa pudica, indicating that the acquisition of essential symbiotic genes may not be sufficient to convert a soil bacterium into a legume symbiont. This proto-rhizobium was evolved through serial cycles of inoculation to M. pudica plantlets and re-isolation of nodule bacteria. In addition to the nod and nif-fix genes involved in Nod Factor synthesis and nitrogen fixation respectively, the pRalta plasmid carries a mutagenesis cassette (imuA2B2C2) that elevates mutation rate when bacteria are free-living in the plant culture medium This transient hypermutagenesis increases the genetic diversity of the rhizospheric bacterial population among which the plant selects the most beneficial variants. FS, frameshift. up112 , up115 , intergenic mutations located 112 bp and 115 bp upstream from the gene indicated
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