Conjugative transfer betweenRhizobium etliendosymbionts inside the root nodule

Abstract

Summary 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.

Experimental Evolution of Legume Symbionts: What Have We Learnt?

“…Horizontal gene transfer (HGT) of symbiotic genes is frequent in nature [113,114], which is consistent with the observations that rhizosphere and nodule environments promote HGT [85,115]. Given that rhizobia co-exist in the soil, and even in nodules, with many other different bacteria [116,117], the transfer of symbiosis genes to new bacterial genera must be extremely common.…”

“…FS, frameshift. up 112 , up 115 , intergenic mutations located 112 bp and 115 bp upstream from the gene indicated.…”

Exploring the evolutionary dynamics of Rhizobium plasmids through bipartite network analysis

“…Additionally, our conclusion is consistent with previous finding in the reference R . etli CFN42 genome, where the plasmids p42b (PUC 6), p42c (PUC 1), p42e (PUC 2) and p42f (PUC 3) are strongly related functionally and have co‐evolved with the chromosomes over a long period, while p42a (PUC 15) and p42d_Sym (PUC 8) were only recently acquired (González et al ., ; Pérez Carrascal et al ., ; Bañuelos‐Vazquez et al ., ). Increasing evidence has been mounting regarding the accessory plasmids' influence on legume‐rhizobia symbiosis under stress, such as resistance to heat, acid, antibiotics, heavy metals, pesticides and oxidative stress protection (Kurchak et al ., ; Streit et al ., , 234; Anjum et al ., ; Vercruysse et al ., ; Naamala et al ., ).…”

Defining the requirements for the conjugative transfer of Rhizobium leguminosarum plasmid pRleVF39b

“…It could be possible that these genes are functional when pRleVF39b conjugates within the rhizospere. A study by Bañuelos-Vázquez et al [37] used donor rhizobia carrying a red fluorescent protein tag in their chromosome to monitor the in situ conjugation of a green fluorescent protein tagged plasmid. A similar method could be utilized to monitor the conjugative functions of the transfer genes of pRleVF39b in the rhizospere.…”