An in vivo whole-plant experimental system for the analysis of gene expression in extraradical mycorrhizal mycelium

Arbuscular mycorrhizal fungi (AMF) establish beneficial mutualistic symbioses with land plants, receiving carbon in exchange for mineral nutrients absorbed by the extraradical mycelium (ERM). With the aim of obtaining in vivo produced ERM for gene expression analyses, a whole-plant bi-dimensional experimental system was devised and tested with three host plants and three fungal symbionts. In such a system, Funneliformis mosseae in symbiosis with Cichorium intybus var. foliosum, Lactuca sativa, and Medicago sativa produced ERM whose lengths ranged from 9.8 ± 0.8 to 20.8 ± 1.2 m per plant. Since ERM produced in symbiosis with C. intybus showed the highest values for the different structural parameters assessed, this host was used to test the whole-plant system with F. mosseae, Rhizoglomus irregulare, and Funneliformis coronatus. The whole-plant system yielded 1–7 mg of ERM fresh biomass per plant per harvest, and continued producing new ERM for 6 months. Variable amounts of high-quality and intact total RNA, ranging from 15 to 65 μg RNA/mg ERM fresh weight, were extracted from the ERM of the three AMF isolates. Ammonium transporter gene expression was successfully determined in the cDNAs obtained from ERM of the three fungal symbionts by RT-qPCR using gene-specific primers designed on available (R. irregulare) and new (F. mosseae and F. coronatus) ammonium transporter gene sequences. The whole-plant experimental system represents a useful research tool for large production and easy collection of ERM for morphological, physiological, and biochemical analyses, suitable for a wide variety of AMF species, for a virtually limitless range of host plants and for studies involving diverse symbiotic interactions.