The Common Bean miR172c microRNA, a Relevant Regulator of the N-Fixing Symbiosis, Is Activated by SPL and AGL/MADS-Domain Transcription Factors.

Nitrogen-fixing symbiosis (NFS) between rhizobia and legume plants is a complex and tightly regulated process. Modules of microRNAs (miRNAs) and their targets from different legumes, such as miR156-SPL and miR172-AP2-1, form part of complex cascades relevant for the regulation of NFS. Based on our previous analysis of the regulatory role of the common bean (Phaseolus vulgaris) miR172c-AP2-1 module, as well as in reports from other legumes about the transcriptional regulation of MIR172 in the NFS, in this work, we demonstrated that PvMIR172c is transcriptionally activated by PvSPL9 and PvFUL-like transcription factors from the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE and AGL (AGAMOUS-LIKE/MADS domain) families, respectively. Bioinformatic analysis of the PvMIR172c gene promoter revealed statistically enriched binding sites for both SPL and AGL transcription factors. The PvSPL9 and PvFUL-like genes are highly expressed in roots/nodules from inoculated plants as compared to roots from fertilized plants. Effector/reporter assays in Nicotiana benthamiana leaves transiently transformed with fluorescent reporter constructs confirmed that PvSPL9d and PvFUL-like activate PvMIR172c expression. In addition, we showed an increased level of miR172c in common bean transgenic roots and nodules overexpressing PvSPL9d or PvFUL-like, while the opposite results held in silenced transgenic tissues. These findings provide an additional understanding of the complex regulatory network controlling NFS in common bean plants.