Conjugative transfer between Rhizobium etli endosymbionts inside the root nodule

Since the discovery that biological nitrogen fixation ensues in nodules resulting from the interaction of rhizobia with legumes, nodules were thought to be exclusive for hosting nitrogen-fixing and plant growth promoting bacteria. In this work, we uncover a novel function of nodules, as a niche permissive to acquisition of plasmids via conjugative transfer. We used Rhizobium etli CFN42, which nodulates Phaseolus vulgaris. The genome of R. etli CFN42 contains a chromosome and six plasmids. pRet42a is a conjugative plasmid regulated by Quorum-Sensing (QS), and pRet42d is the symbiotic plasmid. Here, using confocal microscopy and flow cytometry, we show that pRet42a transfers on the root's surface, and unexpectedly, inside the nodules. Conjugation still took place inside nodules, even when it was restricted on the plant surface by placing the QS traI regulator under the promoter of the nitrogenase gene, which is only expressed inside the nodules, or by inhibiting the QS transcriptional induction of transfer genes with a traM antiactivator on an unstable vector maintained on the plant surface and lost inside the nodules. These results conclusively confirm the occurrence of conjugation in these structures, defining them as a protected environment for bacterial diversification.