Medicago truncatula mycorrhizal roots and Lotus japonicus mycorrhizal and non-mycorrhizal roots, obtained using the sandwich method, were cut into about 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, the ﬁxative solution was changed and the root samples were incubated at 4 °C overnight.
Roots were subsequently dehydrated in a graded series of ethanol (50 %, 70 %, 90 %, 100 % twice) and then put in Neoclear (Merck), with each step on ice for 30 min Neoclear was gradually replaced with parafﬁn (Paraplast Plus; Sigma). In detail, about 10–20 Paraplast Plus chips were added to 20 ml of fresh Neoclear and samples were left for 2 h at room temperature and then for 3 h at 58 °C. Once the chips had dissolved at 58 °C, the mixture was replaced with molten Paraplast Plus at 58 °C. After a first step O.N., paraffin 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 4 °C.
A Leica AS LMD system was used to collect arbuscule-colonized cortical cells from parafﬁn root sections, as described by Balestrini et al. (2007). To follow fungal gene expression, 2500 arbuscule-containing cells (ARB) from M. truncatula roots were microdissected using LMD cells for each of the two biological replicates; RNA was extracted following the Pico Pure kit (Arcturus Engineering) protocol. A DNAse treatment was performed using an RNA-free DNase Set (Qiagen) in Pico Pure column, according to the manufacturer’s instructions (10 ml DNase I + 30 ml RDD buffer for 20 min).
By contrast, to follow the expression of plant genes in different cell-type populations we isolated 1500 Lotus cells of the three different considered populations (ARB, cortical cells containing arbuscule; MNM, non-mycorrhizal cortical cells from a mycorrhizal roots; C, cortical cells from a non-mycorrhizal roots) and, after RNA extraction with the Pico Pure, RNA was subjected to a DNase treatment using a RNase-free DNase (Promega Corp., Madison, WI, U.S.A.; Balestrini et al., 2007). In both the experiments, we used non-amplified RNA samples for qRT-PCR of selected gene candidates.
RNA samples were isolated from the considered cell type populations and directly used in real-time RT-PCR using a specific Bio-Rad kit. We decided to use non-amplified RNA in order to avoid technical artifacts during the RNA amplification. Quantitative RT-PCR amplification reactions, performed with an iCycler apparatus (Bio-Rad), were carried out in a total volume of 25 µl, containing 2 µl RNA, 12.5 µl 2X SYBR Green RT-PCR Reaction Mix, 0.5 µl of each primer (10 µM) and 0.5 µl of iScript Reverse Transcriptase for One-Step RT-PCR (Bio-Rad). The following PCR program was used: 50 °C for 10 min, 95 °C for 5 min, 50 cycles of 95 °C for 10 sec, 60 °C for 30 sec.
A melting curve (80 steps with a heating rate of 0.5 °C per 10 sec and a continuous fluorescence measurement) was recorded at the end of each run to exclude that the primers had generated non-specific PCR products (Ririe et al., 1997). Amplification reactions were successfully performed using specific primers for both fungal and plant genes. Baseline range and Ct values were automatically calculated using the iCycler software. Transcript levels were normalized to the Ct value of a fungal or a plant housekeeping gene (Fiorilli et al., submitted; Giovannetti et al. 2012).
All the reactions were performed on at least two biological and two technical replicates with a good correlation between the replicates. The fact that RNA from cell-type population that contains both fungal and plant RNA (ARB cells) had to be used pure when we used fungal primers, while it was possible to dilute it for plant genes, suggested that the fungus RNA represented a lower proportion than that of the plant.
The use of qPCR-based quantitative analysis allowed us to calculate the percentage of the fungal and plant transcripts in the arbuscule-containing cells, confirming this hypothesis (Fiorilli et al., submitted). We used several fungal and plant genes in this work, confirming the strength of the method. The validity of the results obtained with this method is mostly indicated by the fact that RNA was not amplified before the quantification, and that the exact amount of RNA was preserved in each cell type.