The miR390/TAS3/ARF module governs the expression of an LBD transcription factor to regulate nodulation and root architecture in Medicago truncatula
The microRNA390 targets the TAS3 transcript leading to the production of trans-acting small interference RNAs that subsequently post-transcriptionally repress the mRNAs encoding the Auxin Response Factors (ARF)2, ARF3, and ARF4. This module has been linked to the development of lateral organs in both aerial and underground tissues. Previously, we have shown that the miR390/TAS3/ARFs module mediates the control of lateral roots and symbiotic nodules in legumes. Here, we show that a member of the Lateral Organ Boundaries Domain (LBD) family of transcription factors, designated as MtLBD17/29a, is a target of the miR390/TAS3/ARFs module. Transcriptomic, RT-qPCR and promoter fusion analysis revealed that MtLBD17/29a is induced under symbiotic conditions, whereas no induction was observed in roots overexpressing miR390 or silenced in ARF2/3/4. ChIP-PCR experiments demonstrated that MtARF2 directly binds to an Auxin Response Element (ARE) present in the MtLBD17/29a promoter activating its expression. Knockdown of MtLBD17/29a reduced the length of primary and lateral roots and increasing the density of lateral roots, whereas overexpression of MtLBD17/29a produced the opposite phenotype. Interestingly, both knockdown and overexpression of MtLBD17/29a resulted in a significant reduction in the number of nodules and infection events, as well as impaired the induction of the symbiotic genes Nodulation Signaling Pathway (NSP) 1 and 2, reminiscent of the phenotype observed in ARF2/3/4 silenced roots. These results demonstrate that MtLBD17/29 is regulated by the miR390/TAS3/ARFs module and a direct target of ARF2, revealing a new lateral root regulatory hub recruited by legumes to act in the root nodule symbiotic program. Overall design: RNA-seq libaries were genetated from Medicago truncatula roots overexpressiong the microRNA390 (OX390) and form roots transformed with the empty vector (EV) at 48 hours post-inoculation with water (mock) or with S. meliloti (Sm)