Glomus Titanium Simple
M. truncatula (cv. A17) plants (two trifoliate leaf stage) were grown in pots with 10 plants per pot and inoculated with 1,000 G. intraradices spores per plant. The experiment included three pots (three biological replicates) of mock-inoculated plants and three pots of G. intraradices -inoculated plants. Plants were fertilized with 100 mL/pot of Hoagland solutions containing full strength nitrogen and low Pi (20 microM), twice weekly. Roots were harvested at 27 days post inoculation and frozen in liquid nitrogen. Total RNA was extracted from mycorrhizal roots with Trizol reagent (Invitrogen Corporation, Carlsbad, CA) with additional phenol (pH 6.6): chloroform (1:1, v/v) and chloroform purification steps. RNA from mycorrhizal roots was treated with Turbo DNase I (Ambion Inc., Austin, TX), and column purified with an RNeasy MinElute CleanUp kit (Qiagen). RNA quality and integrity were checked prior to cDNA synthesis using the Bio-Rad Experion analyzer and Experion RNA StdSens analysis kit. Total RNA preparations (three biological replicates) were amplified using the SMART PCR cDNA Synthesis Kit (Clontech) according to the manufacturer's instructions. cDNAs were pooled after purification using QIAquick PCR purification Kit (Qiagen) and used for 454 pyrosequencing.