Auxin Response Factors (ARFs) constitute a large family of transcription factors that mediate auxin-regulated developmental programs in plants. ARF2, ARF3, and ARF4 are post-transcriptionally regulated by the microRNA390 (miR390)/trans-acting small interference RNA 3 (TAS3) module through the action of TAS3-derived trans-acting small interfering RNAs (ta-siRNA). We have previously reported that constitutive activation of the miR390/TAS3 pathway promotes elongation of lateral roots but impairs nodule organogenesis and infection by rhizobia during the nitrogen-fixing symbiosis established between Medicago truncatula and its partner Sinorhizobium meliloti. However, the involvement of the targets of the miR390/TAS3 pathway, i.e., MtARF2, MtARF3, MtARF4a, and MtARF4b, in root development and establishment of the nitrogen-fixing symbiosis remained unexplored. Here, promoter:reporter fusions showed that expression of both MtARF3 and MtARF4a was associated with lateral root development; however, only the MtARF4a promoter was active in developing nodules. In addition, up-regulation of MtARF2, MtARF3, and MtARF4a/b in response to rhizobia depends on Nod Factor perception. We provide evidence that simultaneous knockdown of MtARF2, MtARF3, MtARF4a, and MtARF4b or mutation in MtARF4a impaired nodule formation, and reduced initiation and progression of infection events. Silencing of MtARF2, MtARF3, MtARF4a, and MtARF4b altered mRNA levels of the early nodulation gene nodulation signaling pathway 2 (MtNSP2). In addition, roots with reduced levels of MtARF2, MtARF3, MtARF4a, and MtARF4b, as well as arf4a mutant plants exhibited altered root architecture, causing a reduction in primary and lateral root length, but increasing lateral root density. Taken together, our results suggest that these ARF members are common key players of the morphogenetic programs that control root development and the formation of nitrogen-fixing nodules.

Phaseolus vulgaris plants from the Mesoamerican centre of genetic diversification establish a preferential and more efficient root nodule symbiosis with sympatric Rhizobium etli strains. This is mediated by changes in host gene expression, which might occur either at the transcriptional or at the post-transcriptional level. However, the implication of small RNA (sRNA)mediated control of gene expression in strain selectivity has remained elusive.sRNA sequencing was used to identify host microRNAs (miRNAs) differentially regulated in roots at an early stage of the symbiotic interaction, which were further characterized by applying a reverse genetic approach.In silico analysis identified known and new miRNAs that accumulated to a greater extent in the preferential and more efficient interaction. One of them, designated as Pvu-miR5924, participates in the mechanisms that determine the selection of R. etli strains that will colonize the nodules. In addition, the functional analysis of Pvu-miR390b verified that this miRNA is a negative modulator of nodule formation and bacterial infection.This study not only extended the list of miRNAs identified in P. vulgaris but also enabled the identification of miRNAs that play relevant functions in nodule formation, rhizobial infection and the selection of the rhizobial strains that will occupy the nodule.

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