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Legume plants secrete signaling molecules called flavonoids into the rhizosphere. These molecules activate the transcription of rhizobial nod genes, which encode proteins involved in the synthesis of signaling compounds named Nod factors (NFs). NFs, in turn, trigger changes in plant gene expression, cortical cell dedifferentiation and mitosis, depolarization of the root hair cell membrane potential and rearrangement of the actin cytoskeleton. Actin polymerization plays an important role in apical growth in hyphae and pollen tubes. Using sublethal concentrations of fluorescently labeled cytochalasin D (Cyt-Fl), we visualized the distribution of filamentous actin (F-actin) plus ends in living Phaseolus vulgaris and Arabidopsis root hairs during apical growth. We demonstrated that Cyt-Fl specifically labeled the newly available plus ends of actin microfilaments, which probably represent sites of polymerization. The addition of unlabeled competing cytochalasin reduced the signal, suggesting that the labeled and unlabeled forms of the drug bind to the same site on F-actin. Exposure to Rhizobium etli NFs resulted in a rapid increase in the number of F-actin plus ends in P. vulgaris root hairs and in the re-localization of F-actin plus ends to infection thread initiation sites. These data suggest that NFs promote the formation of F-actin plus ends, which results in actin cytoskeleton rearrangements that facilitate infection thread formation.

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.

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