The Use of Laser Microdissection to Investigate Cell-Specific Gene Expression in Orchid Tissues

  • Raffaella BalestriniEmail author
  • Valeria Fochi
  • Alessandro Lopa
  • Silvia Perotto
Part of the Springer Protocols Handbooks book series (SPH)


In the past decade, laser microdissection (LMD) technology has been widely applied to plant tissues, highlighting the role of different cell-type populations during plant–microbe interactions. In this chapter, a method to apply the LMD approach to study gene expression in specific cell-type populations of orchid mycorrhizal protocorms and roots is described in detail, starting from the preparation of biological material to gene expression analysis by RT-PCR.

Key words

Laser microdissection Gene expression Orchid Serapias vomeracea Tulasnella calospora 



The LMD system upgrade was possible thanks to the financial support of the “Compagnia di San Paolo” (Torino, Italy).


  1. 1.
    Smith SE, Read D (2008) Mycorrhizal Symbiosis, 3rd edn. Academic Press, New YorkGoogle Scholar
  2. 2.
    Leake J (2004) Myco-heterotroph/epiparasitic plant interactions with ectomycorrhizal and arbuscular mycorrhizal fungi. Plant Biol 7:422–428Google Scholar
  3. 3.
    Hynson NA, Weiß M, Preiss K, Gebauer G, Treseder KK (2013) Fungal host specificity is not a bottleneck for the germination of Pyroleae species (Ericaceae) in a Bavarian forest. Mol Ecol 22:1473–1481CrossRefGoogle Scholar
  4. 4.
    Perotto S, Rodda M, Benetti A, Sillo F, Ercole E, Rodda M, Girlanda M, Murat C, Balestrini R (2014) Gene expression in mycorrhizal orchid protocorms suggests a friendly plant–fungus relationship. Planta 239:1337–1349CrossRefGoogle Scholar
  5. 5.
    Zhao M-M, Zhang G, Zhang D-W, Hsiao Y-Y, Guo S-X (2013) ESTs Analysis reveals putative genes involved in symbiotic seed germination in Dendrobium officinale. PLoS One 8:e72705CrossRefGoogle Scholar
  6. 6.
    Liu S-S, Chen J, Li S-C, Zeng X, Meng Z-X, Guo S-X (2015) Comparative transcriptome analysis of genes involved in GA-GID1-DELLA regulatory module in symbiotic and asymbiotic seed germination of Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae). Int J Mol Sci 16:30,190–30,203CrossRefGoogle Scholar
  7. 7.
    Fochi V, Chitarra W, Kohler A, Voyron S, Singan VR, Lindquist EA, Barry KW, Girlanda M, Grigoriev IV, Martin F, Balestrini R, Perotto S (2017) Fungal and plant gene expression in the Tulasnella calosporaSerapias vomeracea symbiosis provides clues about nitrogen pathways in orchid mycorrhizas. New Phytol 213:365–379CrossRefGoogle Scholar
  8. 8.
    Balestrini R, Gómez-Ariza J, Lanfranco L, Bonfante P (2007) Laser Microdissection reveals that transcripts for five plant and one fungal phosphate transporter genes are contemporaneously present in arbusculated cells. Mol Plant Microbe Interact 20:1055–1062CrossRefGoogle Scholar
  9. 9.
    Hogekamp C, Arndt D, Pareira PA, Becker JD, Hohnjec N, Küster H (2011) Laser microdissection unravels cell-type-specific transcription in arbuscular mycorrhizal roots, including CAAT-box transcription factor gene expression correlating with fungal contact and spread. Plant Physiol 157:2023–2043CrossRefGoogle Scholar
  10. 10.
    Hogekamp C, Küster H (2013) A roadmap of cell-type specific gene expression during sequential stages of the arbuscular mycorrhiza symbiosis. BMC Genomics 14:306CrossRefGoogle Scholar
  11. 11.
    Emmert-Buck MR, Bonner RF, Smith PD, Chauqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA (1996) Laser capture microdissection. Science 274:998–1001CrossRefGoogle Scholar
  12. 12.
    Day RC, Grossniklaus U, Macknight RC (2005) Be more specific! Laser-assisted microdissection of plant cells. Trends Plant Sci 10:397–406CrossRefGoogle Scholar
  13. 13.
    Nelson T, Tausta SL, Gandotra N, Liu T (2006) Laser microdissection of plant tissue: what you see is what you get. Annu Rev Plant Biol 57:181–201CrossRefGoogle Scholar
  14. 14.
    Ramsay K, Jones MGK, Wang Z (2006) Laser capture microdissection: a novel approach to microanalysis of plant–microbe interactions. Mol Plant Pathol 7:429–435CrossRefGoogle Scholar
  15. 15.
    Day RC, McNoe LA, Macknight RC (2007) Transcript analysis of laser microdissected plant cells. Technical focus. Physiol Plant 129:267–282CrossRefGoogle Scholar
  16. 16.
    Balestrini R, Bonfante P (2008) Laser microdissection (LM): applications to plant materials. Plant Biosyst 142:331–336CrossRefGoogle Scholar
  17. 17.
    Millar JL, Becker MG, Belmonte MF (2015) Laser microdissection of plant tissues. In: Yeung ECT, Stasolla C, Sumner MJ, Huang BQ (eds) Plant microtechniques and protocols. Springer International Publishing, Switzerland, pp 337–350CrossRefGoogle Scholar
  18. 18.
    Birnbaum K, Shasha DE, Wang JY, Jung JW, Lambert GM, Galbraith DW, Benfey PN (2003) A gene expression map of the Arabidopsis root. Science 302:1956–1960CrossRefGoogle Scholar
  19. 19.
    Galbraith DW, Birnbaum K (2006) Global studies of cell type-specific gene expression in plants. Annu Rev Plant Biol 57:451–475CrossRefGoogle Scholar
  20. 20.
    Asano T, Masumura T, Kusano H, Kikuchi S, Kurita A, Shimada H, Kadowaki K (2002) Construction of specialized cDNA library from plant cells isolated by laser capture microdissection: toward comprehensive analysis of the genes expressed in the rice phloem. Plant J 32:401–408CrossRefGoogle Scholar
  21. 21.
    Kerk NM, Ceserani T, Tausta SL, Sussex IM, Nelson TM (2003) Laser capture microdissection of cells from plant tissues. Plant Physiol 132:27–35CrossRefGoogle Scholar
  22. 22.
    Klink VP, Alkharouf N, MacDonald M, Matthews B (2005) Laser capture microdissection (LCM) and expression analyses of Glycine max (soybean) syncytium containing root regions formed by the plant pathogen Heterodera glycines (soybean cyst nematode). Plant Mol Biol 59:965–979CrossRefGoogle Scholar
  23. 23.
    Inada N, Wildermuth MC (2005) Novel tissue preparation method and cell-specific marker for laser microdissection of Arabidopsis mature leaf. Planta 221:9–16CrossRefGoogle Scholar
  24. 24.
    Cai S, Lashbrook CC (2006) Laser capture microdissection of plant cells from tape transferred paraffin sections promotes recovery of structurally intact RNA for global gene profiling. Plant J 48:628–637CrossRefGoogle Scholar
  25. 25.
    Tang W, Coughlan S, Crane E, Beatty M, Duvick J (2006) The application of laser microdissection to in planta gene expression profiling of the maize anthracnose stalk rot fungus Colletotrichum graminicola. Mol Plant Microbe Interact 19:1240–1250CrossRefGoogle Scholar
  26. 26.
    Balestrini R, Gómez-Ariza J, Klink VP, Bonfante P (2009) Application of laser microdissection to plant pathogenic and symbiotic interaction. J Plant Interact 4:81–92CrossRefGoogle Scholar
  27. 27.
    Gomez SK, Harrison MJ (2009) Laser microdissection and its application to analyze gene expression in arbuscular mycorrhizal symbiosis. Pest Manag Sci 65:504–511CrossRefGoogle Scholar
  28. 28.
    Fiorilli V, Klink VP, Balestrini R (2012) Proteomic analyses of cells isolated by laser microdissection. In: Leung H-C (ed) Integrative proteomics. InTech. ISBN: 978-953-51-0070-6Google Scholar
  29. 29.
    Fang J, Schneider B (2014) Laser microdissection: a sample preparation technique for plant micrometabolic profiling. Phytochem Anal 25:307–313CrossRefGoogle Scholar
  30. 30.
    Gautam V, Sarkar AK (2015) Laser assisted microdissection, an efficient technique to understand tissue specific gene expression patterns and functional genomics in plants. Mol Biotechnol 57:299–308CrossRefGoogle Scholar
  31. 31.
    Nakazono M, Qiu F, Borsuk LA, Schable PS (2003) Laser capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: Identification of genes expressed differentially in epidermal cells or vascular tissue of maize. Plant Cell 15:583–596CrossRefGoogle Scholar
  32. 32.
    Klink VP, Overall CC, Alkharouf NW, MacDonald MH, Matthews BF (2007) Laser capture microdissection (LCM) and comparative microarray expression analysis of syncytial cells isolated from incompatible and compatible soybean (Glycine max) roots infected by the soybean cyst nematode (Heterodera glycines). Planta 226:1389–1409CrossRefGoogle Scholar
  33. 33.
    Ithal N, Recknor J, Nettleton D, Hearne L, Maier T et al (2007) Parallel genome-wide expression profiling of host and pathogen during soybean cyst nematode infection of soybean. Mol Plant Microbe Interact 20:293–305CrossRefGoogle Scholar
  34. 34.
    Hacquard S, Delaruelle C, Legué V, Tisserant E, Kohler A, Frey P, Martin F, Duplessis S (2010) Laser capture microdissection of Uredinia formed by Melampsora larici-populina revealed a transcriptional switch between biotrophy and sporulation. Mol Plant Microbe Interact 23:1275–1286CrossRefGoogle Scholar
  35. 35.
    Chandran D, Inada N, Hather G, Kleindt CK, Wildermuth MC (2010) Laser microdissection of Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and regulators. PNAS 107:460–465CrossRefGoogle Scholar
  36. 36.
    Hacquard S, Tisserant E, Brun A, Legue V, Martin F, Kohler A (2013) Laser microdissection and microarray analysis of Tuber melanosporum ectomycorrhizas reveal functional heterogeneity between mantle and Hartig net compartments. Environ Microbiol 15:1853–1869CrossRefGoogle Scholar
  37. 37.
    Gomez KS, Javot H, Deewatthanawong P, Torres-Jerez Y, Tang I, Blancaflor EB, Udvardi MK, Harrison MJ (2009) Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis. BMC Plant Biol 9:10CrossRefGoogle Scholar
  38. 38.
    Guether M, Neuhäuser B, Balestrini R, Dynowski M, Ludewig U, Bonfante P (2009) A mycorrhizal specific ammonium transporter from Lotus japonicus acquires nitrogen. Plant Physiol 150:73–83CrossRefGoogle Scholar
  39. 39.
    Gaude N, Bortfeld S, Duensing N, Lohse M, Krajinski F (2012) Arbuscule-containing and non-colonized cortical cells of mycorrhizal roots undergo extensive and specific reprogramming during arbuscular mycorrhizal development. Plant J 69:510–528CrossRefGoogle Scholar
  40. 40.
    Balestrini R, Nerva L, Sillo F, Girlanda M, Perotto S (2014) Plant and fungal gene expression in mycorrhizal protocorms of the orchid Serapias vomeracea colonized by Tulasnella calospora. Plant Signal Behav 9:11CrossRefGoogle Scholar
  41. 41.
    Gómez-Ariza J, Balestrini R, Novero M, Bonfante P (2009) Cell-specific gene expression of phosphate transporters in mycorrhizal tomato roots. Biol Fertil Soils 45:845–853CrossRefGoogle Scholar
  42. 42.
    Stasolla C, Yeung EC (2015) Paraffin and polyester waxes. In: Yeung ECT, Stasolla C, Sumner MJ, Huang BQ (eds) Plant microtechniques and protocols. Springer, New York, pp 45–66CrossRefGoogle Scholar
  43. 43.
    Fochi V, Falla N, Girlanda M, Perotto S, Balestrini R (2017) Cell-Specific Expression of Plant Nutrient Transporter Genes in Orchid Mycorrhizae. Plant Sci 263, 39–45CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Raffaella Balestrini
    • 1
    Email author
  • Valeria Fochi
    • 2
  • Alessandro Lopa
    • 2
  • Silvia Perotto
    • 2
    • 1
  1. 1.Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche (CNR)TurinItaly
  2. 2.Dipartimento di Scienze della Vita e Biologia dei SistemiUniversità degli Studi di TorinoTurinItaly

Personalised recommendations