Artificial microRNA (amiR)-mediated silencing of DsRED expression in M. truncatula roots. A) MtErf1 transcripts are specifically induced in mycorrhizal roots (myc) and non- detectable in non-mycorrhizal (nm) roots. MtErf1 and MtEf1 as reference (control) transcripts were amplified from cDNA of mycorrhizal and non-mycorrhizal roots of M. truncatula by RT-PCR. Amplicon sizes are indicated. Two biological replicates are shown. B) Relative levels of MtErf1, MtPt4 and R. irregularis elongation factor 1 alpha (RiEf1)  were determined by qRT-PCR. Values shown are means of 3–4 biological replicates +/− one standard deviation. Each replicate consisted of several roots (n<5) transformed with either the MtPt4pro::amiR-MtErf1 (a) construct or MtPt4pro::uidA control (c). C) amiR-mediated MtErf1 knock-down results in deformed arbuscules. Shown are representative figures of arbuscules observed in roots transformed with either the MtPt4pro:: amiR-MtErf1 construct or the MtPt4pro::uidA control. Bright field images and WGA-Alexafluor 488 fluorescence are shown. Scale bar reesents 25 μm.