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Global and cell-type gene expression profiles in tomato plants colonized by an arbuscular mycorrhizal fungus Valentina Fiorilli1, Marco Catoni2, Laura Miozzi2, Mara Novero1, Gian Paolo Accotto2 and Luisa Lanfranco1 1
Dipartimento di Biologia Vegetale, Universita` degli Studi di Torino, Viale Mattioli 25 10125, Torino, Italy; 2Istituto di Virologia Vegetale, CNR, St. delle
Cacce 73, 10135 Torino, Italy
Summary Author for correspondence: Luisa Lanfranco Tel: 039 011 6705969 Email:
[email protected] Received: 7 April 2009 Accepted: 15 July 2009
New Phytologist (2009) 184: 975–987 doi: 10.1111/j.1469-8137.2009.03031.x
Key words: arbuscular mycorrhizal symbiosis, arbusculated cells, gene expression, Glomus mosseae, laser, microdissection, microarray, tomato.
• Arbuscular mycorrhizal symbiosis develops in roots; extensive cellular reorganizations and specific metabolic changes occur, which are mirrored by local and systemic changes in the transcript profiles. • A TOM2 microarray (c. 12 000 probes) has been used to obtain an overview of the transcriptional changes that are triggered in Solanum lycopersicum roots and shoots, as a result of colonization by the arbuscular mycorrhizal fungus Glomus mosseae. The cell-type expression profile of a subset of genes was monitored, using laser microdissection, to identify possible plant determinants of arbuscule development,. • Microarrays revealed 362 up-regulated and 293 down-regulated genes in roots. Significant gene modulation was also observed in shoots: 85 up- and 337 downregulated genes. The most responsive genes in both organs were ascribed to primary and secondary metabolism, defence and response to stimuli, cell organization and protein modification, and transcriptional regulation. Six genes, preferentially expressed in arbusculated cells, were identified. • A comparative analysis only showed a limited overlap with transcript profiles identified in mycorrhizal roots of Medicago truncatula, probably as a consequence of the largely nonoverlapping probe sets on the microarray tools used. The results suggest that auxin and abscisic acid metabolism are involved in arbuscule formation and ⁄ or functioning.
Introduction One of the most widespread mutualistic associations in nature is the arbuscular mycorrhizal (AM) symbiosis that is formed between soil fungi belonging to Glomeromycota and most land plants (Parniske, 2008). The ability to form this association is widely distributed throughout the plant kingdom, and involves most agricultural, horticultural and hardwood species (Bonfante & Genre, 2008; Smith & Read, 2008). The symbiosis develops in the plant roots where the colonization process occurs in sequential steps that involve both the epidermal and cortical cells. In the root cortex, the fungus develops intercellular hyphae and extensively branched intracellular hyphae called arbuscules (Bonfante et al., 2009). The key functional benefit for both partners is the acquisition of nutrients: the fungus provides the plant with
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mineral nutrients (i.e. phosphate, nitrogen and sulphur) (Govindarajulu et al., 2005; Javot et al., 2007; Allen & Shachar-Hill, 2009) while, in return, it receives carbon compounds from the plant that are essential for the completion of its life cycle (Pfeffer et al., 1999). The symbiosis has a multifunctional nature because AM fungi perform other significant roles, including protection of the plant from biotic and abiotic stress (Pozo & Azco`n-Aguilar, 2007; Aroca et al., 2008). The exploitation of these plantbeneficial symbionts in agro-environments is therefore considered of high environmental relevance and economic value. The establishment of the symbiosis requires complex developmental programmes whose genetic determinants, at least on the plant side, have been described in part through the characterization of mutant lines defective in the colonization process (Reinhardt, 2007; Parniske, 2008).
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Transcriptomic studies, mainly based on microarray technology, have also been instrumental in deciphering the molecular mechanisms that accompany the formation of arbuscular mycorrhizas. In 2003, a 2268-probe cDNA array was used for the first time to monitor changes in transcript abundance along a time course of AM establishment in the model legume Medicago truncatula. The main result was the transient induction of defence-related genes during the early stages of the interaction (Liu et al., 2003). Later, new platforms, representative of a larger portion of the M. truncatula genome, became available (Hohnjec et al., 2005; Liu et al., 2007; Gomez et al., 2009). These studies revealed changes associated with metabolic pathways that control nutritional exchanges, cell wall modifications, secondary metabolism, signal transduction, protein turn-over and transcription (Hohnjec et al., 2005; Liu et al., 2007; Gomez et al., 2009) and allowed several mycorrhiza-responsive genes to be identified. Genome-wide transcript profiling in arbuscular mycorrhizas has been obtained for the monocotyledon Oryza sativa (Gu¨imil et al., 2005) and, more recently, for the legume Lotus japonicus (Guether et al., 2009). Spatial gene expression information is also particularly important in AM symbiosis because a mycorrhizal root is a heterogeneous cell environment that includes colonized and uncolonized cells. The expression patterns specifically associated with arbuscule-containing cells, the key structures of the symbiosis, were explored in tomato (Solanum lycopersicum) for the first time by Balestrini et al. (2007) using laser microdissection technology and, more recently, in the legumes L. japonicus and M. truncatula (Gomez et al., 2009; Guether et al., 2009). To date, transcriptomics studies on arbuscular mycorrhizas have focused on a few plant species, two legumes (Medicago and Lotus) and rice (Oryza sativa), and most of these studies have dealt with changes in transcript levels in roots. Only Liu et al. (2007) monitored local (root) vs systemic (shoot) changes in expression patterns in mycorrhizal Medicago plants. Although tomato is an economically important crop and a model system, with a distinct phylogenetic position and with several resources for genomic research (Shibata, 2005; Barone et al., 2008), global gene expression analyses concerning AM symbiosis have never been undertaken. In this work, the TOM2 microarray platform, which contains c. 12 000 genes (one-third of the whole tomato genome), was used to identify differentially expressed genes in the roots and shoots of S. lycopersicum plants inoculated with Glomus mosseae, an AM fungus widely distributed in agricultural and natural ecosystems. In addition, in order to identify any possible plant determinants of the arbuscule formation, a subset of genes induced in the mycorrhizal roots was selected and their cell-type expression profiles were monitored using laser microdissection (LMD) technology.
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Materials and Methods Biological materials Solanum lycopersicum (L.) (cv. Moneymaker) seeds were surface-sterilized by washing in 70% ethanol with a few drops of Tween 20 for 3 min and in sodium hypochlorite 5% for 13 min, and rinsed three times in distilled water for 10 min. The seeds were placed in agar:H2O (0.6%) in Petri dishes, incubated for 5 d in the dark (25C) and then exposed to light for 4 d. The seedlings were then transferred to pots with sterile quartz sand. Inoculation of Glomus mosseae Gerd. & Trappe BEG12 (Biorize) was performed by mixing the inoculum with sterile quartz sand (30% v ⁄ v). The plants were grown in a growth chamber under a 14 h light (24C) ⁄ 10 h dark (20C) regime, and watered at a rate of 125 ml per plant twice a week with water, and once a week with a modified Long–Ashton solution containing a low phosphorus concentration (3.2 lM Na2HPO4Æ12H2O) (Hewitt, 1966). The plants were harvested 42 d post-inoculation. Portions of the root system from each mycorrhizal plant were selected under a stereomicroscope on the basis of the presence of external mycelium. These root portions were mixed and pooled together and then divided into two samples, one to assess the level of mycorrhiza formation (done over 20 cm of root), and the other for RNA extraction. The mycorrhizal roots were stained with cotton blue and the level of mycorrhiza formation was assessed according to Trouvelot et al. (1986). Only roots showing high percentages for the four parameters considered [frequency of mycorrhiza formation (f %) > 50%, intensity of mycorrhiza formation (M %) > 10%, percentage of arbuscules within infected areas (a %) > 60% and percentage of arbuscules in the root system (A %) > 10%] were used for RNA extraction. RNA extraction and microarray experiment The total RNA was isolated from shoots and roots of single plants with Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. RNA quantity and integrity were examined with Bioanalyzer 2100 (Agilent Technology, Santa Clara, CA, USA). The RNAs were pooled in three biological replicates, each pool containing RNAs from three or four plants (into each pool only one plant with an intensity of mycorrhiza formation < 20% was placed). Pools were prepared in the same way for the shoot and root samples. The TOM2 microarrays were obtained from the Center for Gene Expression Profiles (CGEP; Cornell University, Ithaca, NY, USA). Each microarray contains 11769 oligonucleotide probes designed based on gene transcript sequences from the Lycopersicon Combined Built # 3 unigene database (http://www.sgn.cornell.edu). Three biological replicates were analysed and a ‘dye swap’ app-
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New Phytologist roach was adopted. Total RNA (500 ng) was used to generate direct fluorescently labelled cRNA using the Low RNA Input Linear Amp Kit (Agilent) according to the manufacturer’s instructions. Slides were treated following the prehybridization protocol provided by the manufacturer (http://ted.bti.cornell.edu/). Microarray hybridization was performed using the Gene Expression Hybridization kit (Agilent). Post-hybridization was performed following the manufacturer’s instructions with slight modifications. An additional wash step in 0.05· SSC for 5 min and a dip in absolute ethanol were added before the final quick drying centrifugation. The slides were scanned using an Agilent microarray scanner (G2565BA) at a resolution of 10 lm and laser power set to 90%. The fluorescence data were processed using IMAGENE software (version 5.6; BioDiscovery Inc.; http://www.biodiscovery.com). Normalization and analysis of the microarray data were carried out using LIMMA (BIOCONDUCTOR package) (Smyth, 2005). The values of all the spots on the arrays were per spot and per chip intensitydependent (Lowess) normalized. Significant up- or downregulated genes were filtered for a false discovery rate
