MiR 171 h restricts root symbioses and shows, like its target NSP 2 , a complex transcriptional regulation in Medicago truncatula

Abstract

Background: Legumes 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. Results: We 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. Conclusion: The 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.