MiR171h restricts root symbioses and shows like its target NSP2 a complex transcriptional regulation in Medicago truncatula

Abstract

BackgroundLegumes have the unique capability to undergo root nodule and arbuscular mycorrhizal symbiosis. Both types of root endosymbiosis are regulated by NSP2, which is a target of microRNA171h (miR171h). Although, recent data implies that miR171h specifically restricts arbuscular mycorrhizal symbiosis in the root elongation zone of Medicago truncatula roots, there is limited knowledge available about the spatio-temporal regulation of miR171h expression at different physiological and symbiotic conditions.ResultsWe show that miR171h is functionally expressed from an unusual long primary transcript, previously predicted to encode two identical miR171h strands. Both miR171h and NSP2 transcripts display a complex regulation pattern, which involves the symbiotic status and the fertilization regime of the plant. Quantitative Real-time PCR revealed that miR171h and NSP2 transcript levels show a clear anti-correlation in all tested conditions except in mycorrhizal roots, where NSP2 transcript levels were induced despite of an increased miR171h expression. This was also supported by a clear correlation of transcript levels of NSP2 and MtPt4, a phosphate transporter specifically expressed in a functional AM symbiosis. MiR171h is strongly induced in plants growing in sufficient phosphate conditions, which we demonstrate to be independent of the CRE1 signaling pathway and which is also not required for transcriptional induction of NSP2 in mycorrhizal roots. In situ hybridization and promoter activity analysis of both genes confirmed the complex regulation involving the symbiotic status, P and N nutrition, where both genes show a mainly mutual exclusive expression pattern. Overexpression of miR171h in M. truncatula roots led to a reduction in mycorrhizal colonization and to a reduced nodulation by Sinorhizobium meliloti.ConclusionThe spatio-temporal expression of miR171h and NSP2 is tightly linked to the nutritional status of the plant and, together with the results from the overexpression analysis, points to an important function of miR171h to integrate the nutrient homeostasis in order to safeguard the expression domain of NSP2 during both, arbuscular mycorrhizal and root nodule symbiosis.

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

“…The correlation between NSP1/NSP2 and ARF2/3/4 levels might explain, at least in part, the nodulation and infection phenotypes observed in OX390, MIM390, and ago7 roots. Overexpression of miR171h, which targets and effectively reduces NSP2 transcript levels, also resulted in a reduction of the number of nodules in M. truncatula (Hofferek et al, 2014). The results presented in this work, as well as those described previously by others, led us to propose a model (Fig.…”

“…NIN negatively regulates ENOD11 in the epidermis (Vernié et al, 2015). NF-YA1 is a target of miR169, whereas NSP2 is a target of miR171h (Combier et al, 2006;Hofferek et al, 2014).…”

“…A number of additional transcription factors belonging to the GRAS (Kaló et al, 2005;Smit et al, 2005;Hirsch et al, 2009;Battaglia et al, 2014), Ethylene Response Factor (ERF)/APETALA2 (Andriankaja et al, 2007;Middleton et al, 2007;Cerri et al, 2012), Nuclear factor Y (NF-Y; Zanetti et al, 2010;Laloum et al, 2014;Baudin et al, 2015), and NAC (D'haeseleer et al, 2011) families also are required for nodulation. The expression of some of these transcription factors, such as NF-YA1, Nodulation Signaling Pathway2 (NSP2), and NAC1, is regulated by the action of small regulatory RNAs (sRNAs; Combier et al, 2006;D'haeseleer et al, 2011;De Luis et al, 2012;Lauressergues et al, 2012;Hofferek et al, 2014). Among them, microRNAs (miRNAs) and trans-acting small interference RNAs (tasiRNAs) are endogenous sRNAs of 20 to 22 nucleotides that act as negative regulators of gene expression either by cleaving or inhibiting the translation of their mRNA targets; therefore, they can control developmental processes and the adaptation to changes in environmental conditions.…”

“…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 .…”

Hyphal branching during arbuscule development requires RAM1

“…5O). Nodulation Signaling Pathway1 (NSP1) and NSP2, two GRAS factors that regulate strigolactone biosynthesis (Liu et al, 2011) and influence development of AM in a quantitative manner (Delaux et al, 2013;Hofferek et al, 2014), were likewise not differentially expressed in ram1-3 (Fig. 5, P and Q).…”