Glomus Titanium Simple
For LMD, M. truncatula seedlings were inoculated with G. intraradices using the Millipore sandwich method described by Guether et al. (2009a). After about two months, mycorrhizal roots were dissected into 5 mm pieces and placed in RNase-free tubes containing freshly prepared Farmer's fixative (absolute ethanol/glacial acetic acid 3:1 v/v). After a step under vacuum at room temperature for 20 min, fixative solution was changed and root samples were incubated overnight at 4 C. Roots were then dehydrated in a graded series of ethanol (70, 90, 100 and 100 % in sterilized water) followed by two steps in Neoclear (Merck), with each step on ice for 30 min. Neoclear was gradually replaced with paraffin (Paraplast Plus; Sigma). About 10 Paraplast Plus chips were added to 20 ml of fresh Neoclear and samples were leaved for 2 h at room temperature and then for 3 h at 58 C. Once the chips dissolved at 58 C, the mixture was replaced with molten Paraplast Plus at 58 C, and the medium was changed twice approximately at 4-5 h intervals before embedding in pure paraffin. Root samples were embedded in paraffin in Petri dishes and stored at 4C. A Leica AS LMD (Leica Microsystem, Inc) was used to collect 10,000 arbuscule-colonized cortical cells from paraffin root sections. Total RNA were extracted from microdissected arbuscule-colonized cells using the Pico Pure Kit (Arcturus Engineering). DNase treatment of total RNA was performed using RNAse-free DNase Set (Qiagen). RNA quality and integrity were checked prior to cDNA synthesis using a Bio-Rad Experion analyzer and Experion RNA StdSens analysis kit. Total RNA preparations were amplified using the SMART PCR cDNA Synthesis Kit (Clontech) according to the manufacturer's instructions and used for 454 pyrosequencing.