Transcripts of each selected gene were amplified by RT-PCR. Reactions were carried out in 20 µl PCR mix containing 0.2 U of Taq polymerase and 2 µl 10X buffer with 1.5 mM MgCl2 (Invitrogen, USA), 125 µM dNTPs, 0.5 µM of each gene-specific primer pair (Table 2.3, 2.5 and 2.6), and 1:10 diluted cDNA from spores or roots. Reactions were conducted in a T3000 thermocycler (Biometra, Germany). For the G. mosseae genes, the amplification programme was 60 °C with 33 amplification cycles, and for G. intraradices genes the following program was used: 94 °C for 3 min, 30 cycles (94 °C for 1 min, 58 or 60 °C for 1 min, 72 °C for 1 min), 10 min at 72 °C. PCR products were separated by 1.2 % agarose gel electrophoresis for 25 min at 100 volts, gels were stained 10 min in ethidium bromide and documented under UV light using GelDoc EQ apparatus (BioRad, USA).
Relative quantitative real-time PCR
Relative real-time PCR reactions were carried out to quantify gene transcripts using an ABI PRISM 7900 real-time cycler (Applied BioSystems, Foster City, CA, USA), and the ABsolute SYBR green ROX MIX (ABsoluteTM QPCR® SYBR Green ROX Mix 2x; Thermo Scientific, UK) as fluorescent dye. The gene specific primers are described above (see tables 2.5, 2.6). Each reaction (15 µl) contained 2 µl of 1:20 diluted cDNA from spores and root-extracted RNA, 7.5 µl SYB green mix (from Absolute QPCR SYBR Green kit), 1 pM of each primer and sterile water. The amplification program was performed as follows: 95 °C for 15 min, 40 cycles (95 °C for 15 s, 58 °C for 30 s, 72 °C for 20 s, 75 °C for 15 s). A melting curve (95 °C for 15 s, 55 °C for 15 s, 95 °C for 15 s) was recorded at the end of every run to verify the specificity of amplification. Primer efficiency (E) was calculated using the formula: E=10(-1/slope)-1 (Invitrogen guide for important parameters of quantitative PCR analysis). cDNA amplifications were quantified using 5 cDNA dilutions to produce a linear slope. Three biological repetitions, each with two technical repetitions, were analyzed for each treatment. The threshold was set at 0.2 and Ct (threshold cycle) values were automatically calculated by the SDS 2.3 program (Applied Biosystems, Foster City, USA). To compare to microarray data, the relative expression ratio between mycorrhizal M. truncatula roots and water-activated spores was calculated according to the formula of Pfaffl (2001) using the reference gene GiTEF:
Gene expression was monitored by relative Q-PCR on the Step One Plus Real-Time PCR System Thermal Cycling Block (Applied Biosystems, USA) using the ABsolute SYBR green ROX MIX (ABgene, Epsom, UK), and the gene specific primers described above (see tables 2.5, 2.6). Assays were performed on cDNA from two biological repetitions of differently treated spores and three biological repetitions of mycorrhizal M. truncatula roots for each time point. For each sample, PCR reactions were carried out in technical duplicates using 2 μl of 1:20 diluted cDNA from spores and 1:4 of diluted cDNA from mycorrhizal root as template. PCR was performed as follows: 95 °C for 15 min, 40 cycles (95 °C for 15 s, 58 °C for 30 s, 72 °C for 30 s, 75 °C for 15 s) and for the melting curve: 95 °C for 15 s, 72 °C for 1 min, 95 °C for 15 s. Amplification efficiency of each primer pair was determined using a serial dilution of cDNA. The generated data were analysed by StepOne software v2.1 (Applied Biosystems, USA) using a threshold set at 0.2. The GiTEF gene served as reference, and relative gene expression was calculated using the formula R=2 ΔCT(reference-target).
Absolute quantitative real-time PCR
Transcript abundance of genes corresponding to BEG141_c2781, BEG141_c10704, BEG141_lrc1694 and BEG141_lrc995 was quantified by absolute Q-PCR, using the Step One Plus Real-Time PCR System Thermal Cycling Block (Applied Biosystems, USA) and the ABsolute SYBR green ROX MIX (ABgene, Epsom, UK). The expression of each gene was assayed in two technical repetitions and analyzed by the StepOne software v2.1 (Applied Biosystems, USA). To calculate the absolute number of transcripts present in the original samples, plasmid DNA containing each amplicon was quantified by UV absorbance spectroscopy and linearized by digestion with the restriction enzyme BamH1. A standard curve of each primer pair was determined using a serial dilution of linearized plasmid DNA at dilutions of 10, 102, 103, 104, 105, 106, and 107 copies for each assay. To verify the specificity of each amplification, a melting-curve analysis was included at the end of each PCR run. Relative number of transcripts for each gene was calculated as a ratio to the amount of the reference gene TEF transcripts.
In situ RT-PCR
In situ RT-PCR was performed according to Seddas et al. (2008) on spores from pot cultures or roots prepared as follows. Surface sterilized seeds of M. truncatula line J5 were germinated as described above, and half the seedlings were transplanted into 75-ml pots containing the Terragreen/soil-based G. intraradices BEG141 inoculum mix for inoculated plants. The other half of the seedlings were grown as controls with autoclaved inoculum and 1ml bacterial filtrate for each pot (15ml inoculum in 15ml water filtered with Whatman 2v filter paper). Plants, grown under constant conditions as described above, were harvested from 17 to 21 dai, root systems were washed in ice-cold water and fresh roots were immediately fixed for in situ RT-PCR.
Spores or roots were fixed in 67 % (v / v) ethanol and 23 % (v / v) acetic acid containing 10 % (v / v) DMSO. After 1 h incubation at 4 °C under vacuum, samples were placed in 200 µl fresh fixative solution during 16 h at 4 °C. Samples of fixed materials were washed twice in 67 % (v / v) ethanol and 23 % (v / v) acetic acid then once in DEPC-treated water. Cell walls were permeabilised by digestion with chitinase from Streptomyces griseus (Sigma) and pectinase from Aspergillus niger (Sigma). After washing in the same buffer, samples were digested with proteinase K. Genomic DNA was digested with Hae III (Promega) and Hpa II (Promega) in presence of RNAsin (Promega), then eliminated with DNAse. RNA controls were carried out with RNase before performing the DNAse treatment.
The large ribosomal subunit gene (LSU rRNA) of G. intraradices BEG141 was used to standardize the in situ RT-PCR methodology. Genes putatively encoding two P-type Ca2+ ATPases (BEG141_c2781, BEG141_c10704) and one tonoplast Ca/Mn transporter (2) (BEG141_lrc1694), as well as the control genes DESAT, TEF and GiPT, were chosen to localize gene activities in spores and mycorrhizal roots. Gene-specific primers (Table 2.5 and 2.6) were labeled with Texas Red (MWG-Biotech). Controls were provided by omission of primers in the reverse transcription reaction.
Following amplification, the PCR-mix was removed and samples were post-fixed in 100 % ethanol during 10 min at room temperature then quickly wash in 70 % ethanol. Each sample was deposited on a microscope slide in anti-fading medium (DAKO) and stored at 4 °C in the dark until observation. Fluorescence was observed using a confocal microscope (LEICA TCS SP2 AOBS; Leica Microsystems, Germany). Excitation was carried out with a 594 nm laser used at 27 % of its maximum power with a photomultiplicator at 687 V. The resulting signal was collected between 606 and 640 nm. Autofluorescence of plant and fungal tissues was avoided under these conditions of excitation and signal collection. A 40× oil objective was used to seize images. Each fluorescent image corresponded to the maximum projection of optical sections from a z series, using Leica Confocal software. The resultant depth (z) of each projection was about 300 nm. The optical section number of each projection was between 20 and 40. A Nomarski image was taken in parallel each time.
The same treatment was also done on the 40 day old G. mosseae-inoculated A. sinicus roots (see chapter 2.1.1) to locate gene expression of Gm152. The Gm152 gene-specific primers (Table 2.3) were labeled with TET (Tetrachlorofluorescein, a fluorescent dye labeling) (Sangon Shanghai, China). Fluorescence was observed using a confocal microscope (Zeiss LSM510 META, Germany); a 488 nm laser was used for excitation and the resulting signal was collected at 514 nm for the mycorrhizal root tissues.
Rapid Amplification of cDNA Ends (RACE)
Since the 5′end cDNA sequence of Gm152 was available in the G. mosseas SSH library database, only the 3′end cDNA sequence was obtained using rapid amplification of cDNA ends (RACE) according to Scotto–Lavino et al., 2007. Briefly, to generate the 3’ end, mRNA was reverse transcribed using a primer QI-QO that terminates in two mixed bases (GATC / GAC) followed by 17 Ts and a unique primer sequence. The end was amplified using the primer QO that contains part of this sequence and that binds to these cDNAs at their 3’ends, and a primer that matches the gene of interest, 152GSP1. A second amplification series was then performed using internal primers QI and 152GSP2 to suppress the amplification of non-specific products. Primers were listed in Table 2.7. A LA Taq DNA Polymerase (TaKaRa, Japan) was used for this LA (Long and Accurate) PCR amplification from G. mosseas cDNAs according to the manufacturer’s instructions.
PCR products were visualized by electrophoresis on 1.0% agarose gels stained with ethidium bromide. Bands were excised and purified using the TIANgel Midi Purification Kit (Tiangen Biotech, Beijing, China). PCR products were sequenced (AuGCT Biotechnology, Beijing, China) and homology searches were carried out using NCBI databases by BLAST search. Multiple sequence alignments of translated gene sequences were carried out with the program CLUSTALW (http://www.ebi.ac.uk/clustalW/).
Table of contents :
1.1. The arbuscular mycorrhiza symbiosis
1.2. Molecular mechanisms regulating the AM symbiosis
1.3. Calcium-regulated signaling events in cells
1.4. Thesis objectives
MATERIAL AND METHODS
2.1. Biological materials and growth conditions
2.2. Estimation of mycorrhizal root colonization
2.3. Laser Capture Microdissection (LCM) of arbuscule-containing cortical root cells
2.4. Nucleic acid preparation from spores and roots
2.5. Fungal gene selection and primer design
2.6. Polymerase chain reaction (PCR)
2.7. PCR amplification on genomic DNA
2.8. Yeast complementation
2.9. Statistical analysis
STUDIES OF G. MOSSEAE GENES EXPRESSED BEFORE ROOT CONTACT WITH A. SINICUM
3.1. Fungal gene expression monitored by reverse-transcription (RT)-PCR
3.2. Full length gene sequences and analyses
3.3. Localization of G. mosseae Gm152 gene activity in mycorrhizal roots of A. sinicus
3.4. Discussion and conclusions
GROWTH AND MYCORRHIZA DEVELOPMENT IN WILD-TYPE AND MYCORRHIZADEFECTIVE
4.1. Medicago truncatula wild-type J5 and the mutant TRV25
4.2. Wild type Pisum sativum L. cv Finale and the mutant Pssym36
4.3. Discussion and conclusions
SELECTION OF G. INTRARADICES GENES RELATED TO CALCIUM HOMEOSTASIS AND
SIGNALLING IN ARBUSCULAR MYCORRHIZA INTERACTIONS
5.1. Homology searches and primer design for fungal genes based on selected ESTs in G. intraradices DAOM 197198 (syn. R. irregularis)
5.2. Fungal gene expression monitored by reverse-transcription (RT)-PCR
5.3. Discussion and conclusions
EXPRESSION OF G. INTRARADICES GENES ENCODING CA2+-RELATED PROTEINS IN
INTERACTIONS WITH WILD-TYPE OR MYC- MUTANT ROOTS OF MEDICAGO TRUNCATULA
6.1. Relative quantitative Real-time RT-PCR
6.2. Absolute quantitative Real-time RT-PCR
6.3. Discussion and conclusions
LOCALIZATION OF GENE ACTIVITY IN G. INTRARADICES DURING PRESYMBIOTIC STAGES AND DEVELOPMENT WITH ROOTS
7.1. In situ RT-PCR
7.2. Laser cryo-microdissection
7.3. G. intraradices gene expression in P. sativum L. wild-type and mutant genotypes
7.4. Discussion and conclusions
FUNCTIONAL CHARACTERIZATION OF THREE G. INTRARADICES BEG141 GENES
8.1. Full length gene sequencing and phylogenetic analyses
8.2. Yeast complementation assays
8.3. Discussion and conclusions