Auxin Response Factor 2 (ARF2), ARF3, and ARF4 Mediate Both Lateral Root and Nitrogen Fixing Nodule Development in Medicago truncatula

Auxin Response Factors (ARFs) constitute a large family of transcription factors that mediate auxin-regulated developmental programs in plants. , , and are post-transcriptionally regulated by the microRNA390 (miR390)/ -acting small interference RNA 3 ( ) module through the action of -derived acting small interfering RNAs (ta-siRNA). We have previously reported that constitutive activation of the miR390/ pathway promotes elongation of lateral roots but impairs nodule organogenesis and infection by rhizobia during the nitrogen-fixing symbiosis established between and its partner . However, the involvement of the targets of the miR390/ pathway, i.e., , and , in root development and establishment of the nitrogen-fixing symbiosis remained unexplored. Here, promoter:reporter fusions showed that expression of both and was associated with lateral root development; however, only the promoter was active in developing nodules. In addition, up-regulation of , , and in response to rhizobia depends on Nod Factor perception. We provide evidence that simultaneous knockdown of , and or mutation in impaired nodule formation, and reduced initiation and progression of infection events. Silencing of , and altered mRNA levels of the early nodulation gene nodulation signaling pathway 2 ( ). In addition, roots with reduced levels of , and , as well as 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.