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Quantitative One-Step Protocol to Detect Transcripts in Laser Microdissected Samples

The Arbuscular Mycorrhizal (AM) Symbiosis

Arbuscular mycorrhizal (AM) fungi are an essential feature of the biology and ecology of most terrestrial plants and, as biofertilizers, AM fungi are an emerging issue in many projects focused on low-input agriculture practices. The identification of the events that lead to the formation of an AM, including the mechanisms involved in nutrient transfer, will be a challenging objective for a better exploitation of AMs in agricultural programs. The success of AM fungi in time and space is mainly linked to the nutritional benefits they confer on their plant hosts: they take up inorganic phosphate (Pi) and other macronutrients as well as microelements and water from the soil and deliver them to the plant. The fungus, in turn, receives photosynthetic carbohydrates. Inside the roots, carbohydrates and mineral nutrients are then exchanged across the interface between the plant and the fungus. In addition to their role of biological fertilizers, AM fungi confer resistance to biotic and abiotic stresses and play crucial ecological roles, as they have an impact both on the composition, succession and biodiversity of plant species and on soil structure as a result of improved soil aggregation and increased organic matter (Smith and Read, 2008)


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Arbuscular mycorrhizal root: a heterogeneous cell environment

In the last years the research group at the Plant Protection Institute – CNR (UOS Torino) and Department of Life Science and Systems Biology (University of Torino) has gained good experience in the application of Laser MicroDissection (LMD) to gene expression studies on the AM symbiosis. During intracellular colonization, both partners undergo significant modifications, leading to reciprocal beneficial effects. All of the cellular responses during this process are mirrored by important changes in gene expression, and global transcriptome profiling reveals the genes activated in mycorrhizal roots (reviewed in Küster et al., 2007).

However, whole root expression profiling is complicated by the presence of the multiple cell types that are involved in the interaction. In a fully differentiated mycorrhizal root, in fact, various cell types are present. These cells include colonized epidermal and cortical cells and, among them, non-colonized cells. After the first occurrence of arbuscules, which are considered the key structure of the symbiosis, all developmental stages of the symbiosis are present in the root system. This is due to the fact that in a fully developed symbiosis after the initial colonization of the root cortex, secondary infection events commence and the colonization is reiterated. It is likely that each cell type plays one or more different roles in the interaction (Balestrini et al., 2009). Thus, examination of the process at the molecular level suffers from the drawback that the development of the symbiosis is not a synchronous process. The use of whole organs, containing a mixture of different cell types and stages of arbuscule development, can in fact mask cell type-specific differences in RNA or protein levels.

Laser Microdissection: a powerful tool to dissect the cell complexity in a mycorrhizal root

To overcome this problem, LMD has been used over the last few years to study cell specificity in arbuscular mycorrhizae, and particular attention has been paid to the cortical cells containing the main feature of the symbiosis: the arbuscules. Pioneering work by Balestrini et al., 2007 developed a protocol for harvesting arbuscule-containing cells from paraffin sections of mycorrhizal roots using LMD for the identification of arbuscule-specific phosphate transporter genes in tomato AM roots by a One-Step RT-PCR protocol (Balestrini et al., 2007).

A similar approach was later used to track a limited number of genes in arbuscule-containing cells employing RNA derived from homogenous cell populations (Fiorilli et al., 2009; Gomez-Ariza et al., 2009; Gomez et al., 2009; Guether et al., 2009a; Guether et al., 2009b; Guether et al., 2011), confirming that LMD can be considered a powerful tool for the identification of genes involved during a specific stage of colonization. Interestingly, this approach has also been applied not only to verify the expression of plant genes during the symbiotic stage, but also for fungal genes (Perez-Tienda et al., 2011; Tisserant et al., 2012).

Recently, Hogekamp et al. (2011) combined genome-wide transcriptome profiling based on whole mycorrhizal roots with real-time RT-PCR experiments that relied on characteristic cell types obtained using laser microdissection. In the same year, LMD was also used in combination with high throughput transcriptome analysis in order to investigate the cell type-specific transcriptome reprogramming of root cells during AM development (Gaude et al., 2011).

Real-time PCR on microdissected samples from AM roots

In order to confirm the transcriptional regulation observed by microarray hybridization, real-time RT-PCR has been used on RNA extracted from different cell types isolated by laser microdissection from mycorrhizal and non-mycorrhizal roots (Gaude et al., 2011; Hogekamp et al., 2011), using a two-step protocol on non-amplified material (Gaude et al. 2011) or a one-step protocol on T7-amplified RNA (aRNA; Hogekamp et al., 2011). We have also recently set up a quantitative RT-PCR method (Giovannetti et al., in press; Fiorilli et al., submitted) on RNA extracted from microdissected samples (Figure 1).

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.


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