Abstract
Expression of a mycorrhizal fungal-specific phosphate (P) transporter gene (HcPT1) was studied in mycelium of the ectomycorrhizal fungus Hebeloma cylindrosporum, by in situ reverse transcriptase polymerase chain reaction using amplification of complementary DNA sequences. The expression of HcPT1 was visualised under two different P treatments. Mycelium was transferred to liquid medium with or without P and incubated for 5 days. Under P starvation, mycelium growth and vitality was reduced and the expression of HcPT1 up regulated. Enzyme-labelled fluorescent substrate was used to detect gene expression in situ with epi-fluorescence microscopy and to visualise it at the level of the individual hyphae both in starved and non-starved hyphae. Up-regulation of HcPT1 was observed as a more intense fluorescent signal and from the larger proportion of hyphae that showed expression.
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References
Ausubel FM, Brent R, Kingston RE, Moore D, Siedman JG, Smith JA, Struhl K (1996) Current protocols in molecular biology. 13.7.1–13.7.10. Wiley, New York
Benedetto A, Magurno F, Bonfante P, Lanfranco L (2005) Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae. Mycorrhiza 15:620–627
Bolan NS (1991) A critical review on the role of mycorrhizal fungi in the uptake of phosphorus by plants. Plant Soil 134:189–207
Cellier F, Conéjéro G, Ricaud L, Luu DT, Lepetit M, Gosti F, Casse F (2004) Characterization of AtCHX17, a member of the cation/H+ exchanger, CHX family, from Arabidopsis thaliana suggests a role in K+ homeostasis. Plant J 39:834–846
Chalot M, Javelle A, Blaudez D, Lambilliote R, Cooke R, Sentenac H, Wipf D, Botton B (2002) An update on nutrient transport processes in ectomycorrhizas. Plant Soil 244:165–175
Debaud JC, Gay G (1987) In vitro fruiting under controlled conditions of the ectomycorrhizal fungus Hebeloma cylindrosporum associated with Pinus pinaster. New Phytol 105:429–435
Harrison MJ, van Buuren ML (1995) A phosphate transporter from the mycorrhizal fungus Glomus versiforme. Nature 378:626–629
Harrison MJ, Dewbre GR, Liu J (2002) A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi. Plant Cell 14:2413–2429
Karandashov V, Nagy R, Wegmüller S, Amrhein N, Bucher M (2004) Evolutionary conservation of a phosphate transporter in the arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci USA 101:6285–6290
Koltai H, Bird DM (2000) High throughput cellular localization of specific plant mRNAs by liquid-phase in situ reverse transcription-polymerase chain reaction of tissue sections. Plant Physiol 123:1203–1212
Lambilliotte R, Cooke R, Samson D, Fizames C, Gaymard F, Plassard C, Tatry MV, Berger C, Laudié, Legeai F, Karsenty E, Delseny M, Zimmermann S, Sentenac H (2004) Large-scale identification of genes in the fungus Hebeloma cylindrosporum paves the way to molecular analyses of ectomycorrhizal symbiosis. New Phytol 164:505–513
Maldonado-Mendoza IE, Dewbre GR, Harrison MJ (2001) A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorhizal fungus Glomus intraradices is regulated in response to phosphate in the environment. Mol Plant Microb Interact 14:1140–1148
Martin F, Duplessis S, Ditengou F, Lagrange H, Voiblet C, Lapeyrie F (2001) Developmental cross talking in the ectomycorrhizal symbiosis: signals and communication genes. New Phytol 151:145–154
Morel G, Berger M, Ronsin B, Recher S, Ricard-Blim S, Mertani HC, Lobie PE (1998) In situ reverse transcription-polymerase chain reaction. Applications for light and electron microscopy. Biol Cell 90:137–154
Olsson PA, Van Aarle IM, Allaway WG, Ashford AE, Rouhier H (2002) Phosphorus effects on metabolic processes in monoxenic arbuscular mycorrhiza cultures. Plant Physiol 130:1162–1171
Olsson PA, Hansson MC, Burleigh SH (2006) Effect of P availability on temporal dynamics of carbon allocation and Glomus intraradices high-affinity P transporter gene induction in arbuscular mycorrhiza. Appl Environ Microbiol 72:4115–4120
Rausch C, Daram P, Brunner S, Jansa J, Laloi M, Leggewie G, Amrhein N, Bucher M (2001) A phosphate transporter expressed in arbuscule-containing cells in potato. Nature 414:462–470
Rosewarne GM, Barker SJ, Smith SE, Smith FA, Schachtman DP (1999) A Lycopersicon esculentum phosphate transporter (LePT1) involved in phosphorus uptake from a vesicular–arbuscular mycorrhizal fungus. New Phytol 144:507–516
Scheromm P, Plassard C, Salsac L (1990) Effect of nitrate and ammonium nutrition on the metabolism of the ectomycorrhizal basidiomycete, Hebeloma cylindrosporum Romagn. New Phytol 114:227–234
Tatry MV (2003) Analyse moléculaire des effets bénéfiques de la symbiose ectomycorhizienne sur la nutrition phosphatée de la plante-hôte: identification de deux systèmes de transport de Pi chez le basidiomycète ectomycorhizien Hebeloma cylindrosporum. Ph.D. Thesis, University of Montpellier II, France
Van Aarle IM, Olsson PA, Söderström B (2001) Microscopic detection of phosphatase activity of saprophytic and arbuscular mycorrhizal fungi using a fluorogenic substrate. Mycologia 93:17–24
Acknowledgements
IMvA was funded by an EU Marie-Curie fellowship number QLK5-CT-2002-51566. We would like to thank Dr. Stephanie McInnis (Bristol, UK) for her advice on the protocols related to in situ RT-PCR, Dr. Pål Axel Olsson for critical comments on the manuscript and Dr. Hervé Rouhier for image processing.
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van Aarle, I.M., Viennois, G., Amenc, L.K. et al. Fluorescent in situ RT-PCR to visualise the expression of a phosphate transporter gene from an ectomycorrhizal fungus. Mycorrhiza 17, 487–494 (2007). https://doi.org/10.1007/s00572-007-0127-4
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DOI: https://doi.org/10.1007/s00572-007-0127-4