EXPRESSION OF G. INTRARADICES GENES ENCODING CA2+-RELATED PROTEINS IN INTERACTIONS WITH WILD-TYPE OR MYC- MUTANT ROOTS OF MEDICAGO TRUNCATULA

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Medicago truncatula +Glomus intraradices DAOM197198 (syn. Rhizophagus irregularis Blaszk., Wubet, Renker & Buscot)

Mycorrhizal root material was kindly provided by A. Aloui (INRA, Dijon). Medicago truncatula Gaertn. cv. Jemalong wild-type (line J5) was inoculated with the Glomus intraradices isolate DAOM197198 (syn. Rhizophagus irregularis; Krüger et al., 2012) and plants were grown for 3 weeks according to Aloui et al. (2009). Roots were harvested, washed in ice-cold osmosed water and stored in liquid nitrogen until RNA extraction.
Spores (10,000) of G. intraradices DAOM197198, provided by P. Seddas (INRA, Dijon), were activated for 24h at 25 °C in sterile water and 2 % CO2 (IG150, Jouan incubator) and stored in liquid nitrogen until RNA extraction.

Medicago truncatula + Glomus intraradices BEG141

Wild-type M. truncatula (line J5) and the Myc– dmi3/Mtsym13 mutant TRV25 (provided by G. Duc, INRA-Dijon, France) genotypes were used. The M. truncatula Myc–dmi3/Mtsym13 mutant is mutated for the gene DMI3/MtSYM13 encoding a calcium calmodulin-dependent protein kinase (Lévy et al., 2004). It presents a mycorrhiza-defective phenotype allowing only appressoria formation development of AM colonization (Morandi et al., 2005). The AM fungus Glomus intraradices Smith & Schenck, isolate BEG141, was provided by the International Bank of Glomeromycota (IBG, http://www.ukc.ac.uk/bio/beg/) (INRA-Dijon, France).
Glomus intraradices BEG141 spores were extracted by laceration of colonized leek roots (6-month-old leek pot culture) in sterile water using a blender. Two biological repetitions of spores (5,000) were activated for 24h at 25 °C in water and 2 % CO2 (IG150, Jouan incubator). Root exudates of M. truncatula were collected according to Seddas et al (2009). Briefly, five germinated seedlings of M. truncatula per each well were incubated in 3 ml of sterile water in a six-well plate (Falcon Multiwell, BD Sciences, San Jose, CA, U.S.A.) for 7 days. After elimination of all plant residues, this crude plant root exudates were directly used with spores. Two biological repetitions of spores (5,000) were germinated in wild-type J5 or TRV25 mutant root exudates separately for 6 days and two biological repetitions of 5,000 spores were germinated in water for 6 days as control. For a time-course study of gene expression during mycorrhiza development, surface sterilized seeds (6 min in 98 % sulphuric acid, 10 min in 3 % sodium hypochlorite) of M. truncatula line J5 and the Myc– TRV25 mutant were germinated for 3 days on 0.7 % Bactoagar (Difco Laboratories, Detroit) at 25 °C in the dark and then transplanted into 50-ml or 75-ml pots containing a mixture (2:1, vol / vol) of Terragreen (OilDri-US special, Mettman, Germany) and a neutral clay soil-based inoculum (spores, roots, and hyphae) of G. intraradices BEG141 produced on leek. Inoculum was checked for the absence of rhizobia by growing with the hypernodulating M. truncatula genotype TR122 for 3 weeks. Plants were grown under constant conditions: 355 μE/m2/s, 16h-24 °C and 8h-21 °C, day and night, respectively, 70 % humidity. They were fertilized with 3 ml (50-ml pot) or 5 ml/pot (75-ml pot) of a modified Long Ashton solution (Hewitt et al., 1966) without phosphate, enriched in nitrogen (Table 2.2) twice a week, and received distilled water on other days. Plants were harvested at 4, 6, 17 or 21 days after inoculation (dai) for the J5 genotype, and 4 or 6 dai for the TRV25 mutant. Fresh roots from each harvest were washed in ice-cold water and immediately stored in liquid nitrogen for RNA extraction. At each time point, root systems of additional plants were stained for mycorrhizal root colonization (see below).

Pisum sativum L. + Glomus intraradices BEG141

Wild-type pea (P. sativum L.) cv. Finale and the symbiotic mutant Pssym36 (previously denoted RisNod24; provided by A. Borisov, ARRIAM, St. Petersburg, Russia) were used. The mutant Pssym36 is defective for arbuscule formation (Gianinazzi-Pearson, 1996).
Seeds were surface-disinfected 10-min in a sodium hypochlorite solution (3 %) at room temperature, washed with distilled water and germinated in Petri dishes (12-cm diameter) on humid filter paper at 24 °C in the dark for 5 days. Seedlings were planted into pots containing 200 ml of a 1:1 mixture of quartz sand and mycorrhizal soil-based inoculum (spores, roots, and hyphae) of G. intraradices BEG141. Plants were grown under control conditions (326 μ mol m-2 s -1, 24/22 °C, 16 h light, 70 % relative humidity), fertilized with 10 ml / pot of a modified Long Ashton solution without phosphorus, enriched in nitrogen (Table 2.2) three times a week, or 10 ml of distilled water on other days. Plants were harvested at 21 days after inoculation (dai). Root systems were washed in cold water, root and shoot fresh weights were recorded, and roots were stored in liquid nitrogen for RNA extraction or directly stained to determine the AM fungal development on randomly sampled root pieces (see below).

Estimation of mycorrhizal root colonization

To determine the number of appressoria and total root colonization for M. truncatula, fresh roots were washed under running tap water, cleared for 48 h in 10 % KOH at room temperature and stained overnight with 0.05 % trypan blue in glycerol (4 and 6 dai roots) or 30 min in 10 % KOH at 90 °C and 15 min in 0.05 % trypan blue at 90 °C (17, 21 dai roots). For P. sativum roots were cleared 1 hour at 90°C in 10 % KOH and stained 15 min in 0.05 % trypan blue at 90 °C. Root systems were cut into 1 cm pieces and 30 root fragments/plant were observed microscopically according to Trouvelot et al. (1986) (Fig. 2.2). Mycorrhiza parameters were calculated using the MYCOCALC program (http://www.dijon.inra.fr/mychintec/Mycocalc-prg/download.html). The parameters used were F % (frequency of mycorrhizal colonization in the root system), M % (intensity of mycorrhizal colonization in the root cortex), m % (intensity of mycorrhizal colonization in mycorrhizal root fragments), A % (arbuscule abundance in the root system), a % (arbuscule abundance in mycorrhizal root fragments). Also the development of vesicles in roots was estimated as V % (vesicle abundance in the root system), v % (vesicle abundance in mycorrhizal root fragments), using the same scoring as for arbuscules.

RNA extraction from laser-dissected cells

Harvested laser-dissected cells were incubated in the RNA extraction buffer for 45 min at 42 °C, centrifuged at 800 × g for 2 min, and stored at –80 °C until use. RNA was extracted using the Picopure RNA Isolation Kit according to the manufacturer’s protocol.

One-step RT-PCR

After RNA extraction, the RT-PCR experiments were performed using a One-Step RT-PCR kit (Qiagen, Valencia, CA, U.S.A.). Reactions were carried out in a final volume of 50 μl containing 10 μl of 5× buffer, 2 μl of 10mM dNTPs, 3 μl of each primer (10 μM), 2 μl of One-Step RT-PCR enzyme mix, 8 units of RNAse inhibitor, and 3 ng of total RNA from arbusculated cells and noncolonized cortical cells from mycorrhizal roots. The samples were incubated for 30 min at 58 °C, followed by 15-min incubation at 95 °C. Amplification reactions (the primers and conditions are indicated in Table 2.5 and 2.6) were run for 35 to 40 cycles. The PCR products were separated by agarose gel electrophoresis in a Trisacetate-EDTA 0.5× buffer, stained with ethyl bromide, and visualized using a VersaDoc Imaging System (Bio-Rad Laboratories, CA, U.S.A.). Experiments were repeated using arbusculated cells from two different biological repetitions (C1 and C2) and representative results are shown.

Nucleic acid preparation from spores and roots

Total RNA was extracted from G. mosseae quiescent spores and spores germinated in water or stimulated by plant root exudates (2000 per treatment) using a E.Z.N.A. fungal RNA kit (Omega Bio-Tek Inc, USA) (E.Z.N.A., EaZy Nucleic Acid), and DNA removed by RNase-Free DNase (Omega Bio-Tek Inc, USA) according to the manufacturer’s protocol. RNA quantity was estimated using an Eppendorf biophotometer (Eppendorf, Germany).
Total RNA was extracted from G. intraradices DAOM197198 spores, G. intraradices BEG141 spores and mycorrhizal (G. intraradices BEG141) M. truncatula roots using a SV Total RNA Isolation System (Promega, Madison, WI, USA), according to the manufacturer’s protocol. The DNase-containing columns of the Promega kit for total RNA isolation (SV Total RNA Isolation System) were used to remove any DNA contamination.
Total RNA was isolated from mycorrhizal (G. intraradices DAOM197198) M. truncatula roots by A. Aloui using the phenol-chloroform method according to Franken and Gnadinger (1994). In order to test the specificity of the fungal primers, cDNA from non-mycorrhizal M. truncatula root was used as control; total RNA was extracted from non-mycorrhizal M. truncatula roots using the Qiagen RNeasy Plant Mini Kit (74904) following the manufacturer’s instructions (Qiagen, Hilden, Germany). RNA integrity, quantity and quality were controlled by 1.2 % denaturing agarose gel electrophoresis and photometric analysis (Eppendorp biophotometer).

cDNA synthesis

RNA from G. mosseae quiescent spores (0.030 µg/µl), from spores germinated in water (0.028 μg/μl) and from spores stimulated by crude plant root exudates (0.083 μg/μl) (Eppendorf biophotometer) were used to synthesize cDNA according to the protocol of the Superscript III Reverse Transcriptase kit (Invitrogen, USA). 0.2 μg DNA-free RNA from spores were reverse transcribed with 1 μg oligo(dT)18 (Promega, USA) and 200 U M-MLV Reverse transcriptase RNase H Minus (Promega) according to the Promega protocol. cDNA was amplified via the protocol as follows: 42 °C for 1 h and 70 °C for 2 min and stored at -20 °C until use.
RNA from two batches of spores and three biological repetitions of mycorrhizal M. truncatula roots and non-mycorrhizal M. truncatula were used to synthesize cDNA with Superscript III Reverse Transcriptase according to the Invitrogen protocol. 0.15 μg or 1 μg DNA-free RNA from spores and mycorrhizal roots were separately reverse transcribed into first-strand cDNA. The reaction mix containing RNA, 1 μg oligo(dT)15 (Promega) and 0.5 mM dNTPs (Final concentration) was preheated at 70 °C for 5 min and cooled on ice for 3 min. 40 U RNasine RNA inhibitor (Promega), 300 U M-MLV Reverse transcriptase RNase H Minus (Promega) and reaction buffer were added to obtain a final mix volume of 25 μl and amplified using the following programme: 25 °C for 15 min, 42 °C for 1 h and 96 °C for 2 min. cDNA was stored at -20 °C until use.

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Genomic DNA extraction from fungal spores

G. mosseae genomic DNA was extracted from 2000 spores using the protocol described by Zézé et al. (1994). Briefly, spores were gently crushed in TE buffer containing 2 % mercaptoethanol, the suspension was centrifuged and nuclei were resuspended in lysis buffer. After centrifugation, the supernatant was collected and proteins were precipitated with potassium acetate. Nucleic acids contained in the supernatant were treated with DNAse-free RNAse A, and then purified with phenol-chloroform. The purified DNA was precipitated in ethanol at -70 °C, centrifuged, washed with 70 % ethanol, air dry and resuspended in TE buffer or sterile water.

Plasmid DNA preparation

Amplified fragments of cDNA (see below) were cloned into a plasmid vector using the TOPO TA Cloning kit (Invitrogen Corporation, Carlsbad, CA, USA). The plasmids were recuperated by white-blue selection and PCR. Recombinants (white colonies) were multiplied in liquid LB culture containing ampicillin (50 μg / ml), and plasmid DNA was isolated using the NucleoSpin®Plasmid kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions. The yield and purity of plasmid DNA were estimated using an Eppendorp BioPhotometer. Cloned PCR products were sent for sequencing to MWG-Biotech (Ebersberg, Germany).

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 :

CHAPTER 1 GENERAL INTRODUCTION
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
CHAPTER 2 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
CHAPTER 3 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
CHAPTER 4 GROWTH AND MYCORRHIZA DEVELOPMENT IN WILD-TYPE AND MYCORRHIZADEFECTIVE MUTANT PLANTS
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
CHAPTER 5 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
CHAPTER 6 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
CHAPTER 7 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
CHAPTER 8 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
CHAPTER 9 GENERAL DISCUSSION AND CONCLUSIONS
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