Overexpression of miR160 affects root growth and nitrogen-fixing nodule number in Medicago truncatula

Abstract

Auxin action is mediated by a complex signalling pathway involving transcription factors of the auxin response factor (ARF) family. In Arabidopsis, microRNA160 (miR160) negatively regulates three ARF genes (ARF10/ARF16/ARF17) and therefore controls several developmental processes, including primary and lateral root growth. Here, we analysed the role of miR160 in root development and nodulation in Medicago truncatula Gaertn. Bioinformatic analyses identified two main mtr-miR160 variants (mtr-miR160abde and mtr-miR160c) and 17 predicted ARF targets. The miR160-dependent cleavage of four predicted targets in roots was confirmed by analysis of parallel analysis of RNA ends (PARE) data and RACE-PCR experiments. Promoter-GUS analyses for mtr-miR160d and mtr-miR160c genes revealed overlapping but distinct expression profiles during root and nodule development. In addition, the early miR160 activation in roots during symbiotic interaction was not observed in mutants of the nodulation signalling or autoregulation pathways. Composite plants that overexpressed mtr-miR160a under two different promoters exhibited distinct defects in root growth and nodulation: the p35S:miR160a construct led to reduced root length associated to a severe disorganisation of the RAM, whereas pCsVMV:miR160a roots showed gravitropism defects and lower nodule numbers. Our results suggest that a regulatory loop involving miR160/ARFs governs root and nodule organogenesis in M. truncatula.

The Root Hair “Infectome” of Medicago truncatula Uncovers Changes in Cell Cycle Genes and Reveals a Requirement for Auxin Signaling in Rhizobial Infection

“…Although the function of the putative repressor ARF16a has not been studied directly in legumes, the effect of overexpressing miR160 (which targets ARF16 as well as other closely related ARFs) has been examined in soybean and M. truncatula during nodulation. Overexpression of miR160 in soybean did not affect infections in root hairs; in a similar M. truncatula study, infections were not scored (Bustos-Sanmamed et al, 2013;Turner et al, 2013). The apparent discrepancy between our work and the soybean study could be due to inefficient knockdown of ARF16 by miR160 or the differential regulation of auxin in determinate versus indeterminate nodulation (Subramanian et al, 2006Wasson et al 2006).…”

Soybean miR172c Targets the Repressive AP2 Transcription Factor NNC1 to Activate ENOD40 Expression and Regulate Nodule Initiation

“…] legume partners are to determine how the association arises, how nodule organogenesis and thus nodule number are regulated, and how nitrogen fixation is controlled. Recently, there has been a great deal of interest in understanding the roles of miRNAs during nodulation in legumes Bazin et al, 2012;Bustos-Sanmamed et al, 2013;Turner et al, 2013). Our analysis here identified miR172c-NNC1 as a critical regulatory module of nodulation that targets early nodulin ENOD40 genes and is essential for nodule initiation and nodule organogenesis in soybean.…”

The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth

“…However, the roles played by these miRNAs in the control of nodulation have been investigated only for a few of them. In M. truncatula, overexpression of miR160 (Bustos-Sanmamed et al, 2013), miR164 (D'haeseleer et al, 2011), miR166 (Boualem et al, 2008), miR169 (Combier et al, 2006), and miR171h (Hofferek et al, 2014), which target the AUXIN RESPONSE FACTOR (ARF)10/16/17, NAC1, HD-Zip, NF-YA1, and NSP2 transcription factors, respectively, negatively affected nodule number and/or development. miR390 is an evolutionarily conserved 21-nucleotide miRNA that targets the Trans-Acting Short Interference RNA3 (TAS3) transcript in two different sites, a mode of action referred to as the two-hit model and illustrated in Figure 1A .…”