, Volume 20, Issue 6, pp 427–443 | Cite as

Symbiosis-related pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices

  • Elena Kuznetsova
  • Pascale M. A. Seddas-Dozolme
  • Christine Arnould
  • Marie Tollot
  • Diederik van Tuinen
  • Alexey Borisov
  • Silvio Gianinazzi
  • Vivienne Gianinazzi-PearsonEmail author
Original Paper


The arbuscular mycorrhiza association results from a successful interaction between genomes of the plant and fungal symbiotic partners. In this study, we analyzed the effect of inactivation of late-stage symbiosis-related pea genes on symbiosis-associated fungal and plant molecular responses in order to gain insight into their role in the functional mycorrhizal association. The expression of a subset of ten fungal and eight plant genes, previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated wild-type and isogenic genotypes of pea mutated for the PsSym36, PsSym33, and PsSym40 genes where arbuscule formation is inhibited or fungal turnover modulated, respectively. Microdissection was used to corroborate arbuscule-related fungal gene expression. Molecular responses varied between pea genotypes and with fungal development. Most of the fungal genes were downregulated when arbuscule formation was defective, and several were upregulated with more rapid fungal development. Some of the plant genes were also affected by inactivation of the PsSym36, PsSym33, and PsSym40 loci, but in a more time-dependent way during root colonization by G. intraradices. Results indicate a role of the late-stage symbiosis-related pea genes not only in mycorrhiza development but also in the symbiotic functioning of arbuscule-containing cells.


Glomus intraradices Pisum sativum Symbiosis-related plant mutants Gene expression Laser microdissection 



This work was supported by the French Foreign Affairs Ministry (doctoral grant to E. Kuznetsova), the Regional Council of Burgundy (Project no. 07 9201 AAO40 S3623), RFBR (10-04-01026, 10-04-01146), and the Russian Ministry of Science and Education (02.512.11.2254). Authors thank G. Duc for seeds of P. sativum cv. Finale and Pssym36 pea genotypes, C. Desmetz and Z. Hao for assistance respectively in RNA control and statistical analyses, and A. Colombet and V. Monfort for technical assistance.


  1. Aono T, Maldonado-Mendoza IE, Dewbre GR, Harrison MJ, Saito M (2004) Expression of alkaline phosphatase genes in arbuscular mycorrhizas. New Phytol 162:525–534CrossRefGoogle Scholar
  2. Balestrini R, Lanfranco L (2006) Fungal and plant gene expression in arbuscular mycorrhizal symbiosis. Mycorrhiza 16:509–524CrossRefPubMedGoogle Scholar
  3. Balestrini R, Berta G, Bonfante P (1992) The plant nucleus in mycorrhizal roots: positional and structural modifications. Biol Cell 75:235–243CrossRefGoogle Scholar
  4. Balestrini R, Gomez-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 20:1055–1062CrossRefPubMedGoogle Scholar
  5. Bonfante-Fasolo P, Scannerini S (1992) The cellular basis of plant–fungus interchanges in mycorrhizal associations. In: Allen MF (ed) Mycorrhizal functioning: an integrative plant–fungal process. Chapman and Hall, New York, pp 65–101Google Scholar
  6. Borisov AY, Voroshilova VA, Zhukov VA, Zhernakov AI, Danilova TN, Shtark OY, Naumkina TS, Tsyganov VE, Madsen LH, Sanjuan J, Olivares J, Priefer UB, Ellis N, Stougaard J, Tikhonovich IA (2004) Pea (Pisum sativum) regulatory genes controlling development of nitrogen-fixing nodules and arbuscular mycorrhiza. In: Tikhonovich I, Lugtenberg B, Provorov N (eds) Biology of plant–microbe interactions, vol 4. IS-MPMI, St. Paul, pp 502–505Google Scholar
  7. Brechenmacher L, Weidmann S, van Tuinen D, Chatagnier O, Gianinazzi S, Franken P, Gianinazzi-Pearson V (2004) Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula–Glomus mosseae interactions. Mycorrhiza 14:253–262CrossRefPubMedGoogle Scholar
  8. Breuninger M, Requena N (2004) Recognition events in AM symbiosis: analysis of fungal gene expression at the early appressorium stage. Fungal Gen Biol 41:794–804CrossRefGoogle Scholar
  9. Bucher M (2007) Functional biology of plant phosphate uptake at root and mycorrhiza interfaces. New Phytol 173:11–26CrossRefPubMedGoogle Scholar
  10. Dickson S (2004) The Arum–Paris continuum of mycorrhizal symbioses. New Phytol 163:187–200CrossRefGoogle Scholar
  11. Drissner D, Kunze G, Callewaert N, Gehrig P, Tamasloukht M, Boller T, Felix G, Amrhein N, Bucher M (2007) Lyso-phosphatidylcholine is a signal in the arbuscular mycorrhizal symbiosis. Science 318:265–268CrossRefPubMedGoogle Scholar
  12. Duc G, Trouvelot A, Gianinazzi-Pearson V, Gianinazzi S (1989) First report of non-mycorrhizal plant mutants (Myc−) obtained in pea (Pisum sativum L.) and fababean (Vicia faba L.). Plant Sci 60:215–222CrossRefGoogle Scholar
  13. Engvild KC (1987) Nodulation and nitrogen fixation mutants of pea, Pisum sativum. Theor Appl Genet 74:711–713CrossRefGoogle Scholar
  14. Franken P, Gnädinger F (1994) Analysis of parsley arbuscular endomycorrhiza: infection development and mRNA level of defense-related genes. Mol Plant–Microbe Interact 7:612–620Google Scholar
  15. Frenzel A, Manthey K, Perlick AM, Meyer F, Pülher A, Krajinski F, Küster H (2005) Combined transcriptome profiling reveals a novel family of arbuscular mycorrhizal-specific Medicago truncatula lectin genes. Mol Plant–Microbe Interact 18:771–782CrossRefPubMedGoogle Scholar
  16. Genre A, Bonfante P (2002) Epidermal cells of a symbiosis-defective mutant of Lotus japonicus show altered cytoskeleton organisation in the presence of a mycorrhizal fungus. Protoplasma 219:43–50CrossRefPubMedGoogle Scholar
  17. Genre A, Chabaud M, Faccio A, Barker DG, Bonfante P (2008) Prepenetration apparatus assembly precedes and predicts the colonization patterns of arbuscular mycorrhizal fungi within the root cortex of both Medicago truncatula and Daucus carota. Plant Cell 20:1407–1420CrossRefPubMedGoogle Scholar
  18. Gianinazzi-Pearson V (1996) Plant cell responses to arbuscular mycorrhizal fungi: getting to the roots of the symbiosis. Plant Cell 8:1871–1883CrossRefPubMedGoogle Scholar
  19. Gianinazzi-Pearson V, Gianinazzi S, Guillemin JP, Trouvelot A, Duc G (1991) Genetic and cellular analysis of resistance to vesicular arbuscular (VA) mycorrhizal fungi in pea mutants. In: Hennecke H, Verma DPS (eds) Advances in molecular genetics of plant–microbe interactions. Kluwer, The Netherlands, pp 336–342Google Scholar
  20. Gianinazzi-Pearson V, Gollotte A, Lherminier J, Tisserant B, Franken P, Dumas-Gaudot E, Lemoine M-C, van Tuinen D, Gianinazzi S (1995) Cellular and molecular approaches in the characterization of symbiotic events in functional arbuscular mycorrhizal associations. Can J Bot 73:S526–S532CrossRefGoogle Scholar
  21. Gianinazzi-Pearson V, Dumas-Gaudot E, Gollotte A, Tahiri-Alaoui A, Gianinazzi S (1996) Cellular and molecular defence-related root responses to invasion by arbuscular mycorrhizal fungi. New Phytol 133:45–57CrossRefGoogle Scholar
  22. Gianinazzi-Pearson V, Armould C, Oufattole M, Arango M, Gianinazzi S (2000) Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco. Planta 211:609–613CrossRefPubMedGoogle Scholar
  23. Gianinazzi-Pearson V, Séjalon-Delmas N, Genre A, Jeandroz S, Bonfante P (2007) Plants and arbuscular mycorrhizal fungi: cues and communication in the early steps of symbiotic interactions. Adv Bot Res 46:181–219CrossRefGoogle Scholar
  24. Gomez SK, Javot H, Deewatthanawong P, Torres-Jerez I, Tang Y, 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 9:10CrossRefPubMedGoogle Scholar
  25. Groysman M, Hornstein I, Alcover A, Katzav S (2002) Vav1 and Ly-GDI, two regulators of Rho GTPases, function cooperatively as signal transducers in T cell antigen receptor-induced pathways. J Biol Chem 277:50121–55013CrossRefPubMedGoogle Scholar
  26. Grunwald U, Nyamsuren O, Tamasloukht M, Lapopin L, Becker A, Mann P, Gianinazzi-Pearson V, Krajinski F, Franken P (2004) Identification of mycorrhiza-regulated genes with arbuscule development-related expression profile. Plant Mol Microbiol 55:553–566Google Scholar
  27. 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 14:2413–2429CrossRefPubMedGoogle Scholar
  28. Hewitt EJ (1966) Sand and water culture methods used in the study of plant nutrition. Technical communication no. 22 (revised 2nd edition) of the Commonwealth Bureau of Horticulture and Plantation Crops, East Malling, Maidstone. Kent. Commonwealth Agricultural Bureaux, Farnham Royal, p 547Google Scholar
  29. Hohnjec N, Vieweg MF, Pühler A, Becker A, Küster H (2005) Overlaps in the transcriptional profiles of Medicago truncatula roots inoculated with two different Glomus fungi provide insights into the genetic program activated during arbuscular mycorrhiza. Plant Physiol 137:1283–1301CrossRefPubMedGoogle Scholar
  30. Jacobi LM, Petrova OS, Tsyganov VE, Borisov AY, Tikhonovich IA (2003a) Effect of mutations in the pea genes Sym33 and Sym40. I. Arbuscular mycorrhiza formation and function. Mycorrhiza 13:3–7PubMedGoogle Scholar
  31. Jacobi LM, Zubkova LA, Barmicheva EM, Tsyganov VE, Borisov AY et al (2003b) Effects of mutations in the pea genes Sym33 and Sym40. II. Dynamics of arbuscule development and turnover. Mycorrhiza 13:9–16PubMedGoogle Scholar
  32. Janoušková M, Seddas P, Mrnka L, van Tuinen D, Dvořáčková A, Gianinazzi-Pearson V, Vosátka M, Gollotte A (2009) Development and activity of Glomus intraradices as affected by coexistence with Glomus claroideum in one root system. Mycorrhiza 19:393–402CrossRefPubMedGoogle Scholar
  33. Javot H, Penmetsa V, Terzaghi N, Cook DR, Harrison MJ (2007) A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci USA 104:1720–1725CrossRefPubMedGoogle Scholar
  34. Jonak C, Ligterink W, Hirt H (1999) MAP kinases in plant signal transduction. Cell Mol Life Sci 55:204–213CrossRefPubMedGoogle Scholar
  35. Journet EP, van Tuinen D, Gouzy J, Crespeau H, Carreau V, Farmer MJ, Niebel A, Schiex T, Jaillon O, Chatagnier O, Godiard L, Micheli F, Kahn D, Gianinazzi-Pearson V, Gamas P (2002) Exploring root symbiotic programs in the model legume Medicago truncatula using EST analysis. Nucl Acids Res 30:5579–5592CrossRefPubMedGoogle Scholar
  36. Kojima T, Hayatsu M, Saito M (2001) Electrophoretic detection and partial purification of the phosphatase specific for arbuscular mycorrhizal symbiosis. Bull Nat Grassland Res Inst 60:9–11Google Scholar
  37. Kosuta S, Chabaud M, Lougnon G, Gough C, Dénarié J (2003) A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-specific MtENOD expression in roots of Medicago truncatula. Plant Physiol 131:952–962CrossRefPubMedGoogle Scholar
  38. Krajinski F, Hause B, Gianinazzi-Pearson V, Franken P (2002) Mtha1, a plasma membrane H+-ATPase gene from Medicago truncatula, shows arbuscule-specific induced expression in mycorrhizal tissue. Plant Biol 4:754–761CrossRefGoogle Scholar
  39. Küster H, Hohnjec N, Krajinski F, El Yahyaoui F, Manthey K, Gouzy J, Dondrup M, Meyer F, Kalinowski J, Brechenmacher L, van Tuinen D, Gianinazzi-Pearson V, Pühler A, Gamas P, Becker A (2004) Construction and validation of cDNA-based Mt6k-RIT macro- and microarrays to explore root endosymbioses in the model legume Medicago truncatula. J Biotechnol 108:95–113CrossRefPubMedGoogle Scholar
  40. Lanfranco L, Novero M, Bonfante B (2005) The mycorrhizal fungus Gigaspora margarita possesses a CuZn superoxide dismutase which is up-regulated during the symbiosis with legume hosts. Plant Physiol 137:1319–1330CrossRefPubMedGoogle Scholar
  41. Lapopin L, Gianinazzi-Pearson V, Franken P (1999) Comparative differential display analysis of arbuscular mycorrhiza in Pisum sativum and a mutant defective in late stage development. Plant Mol Biol 41:669–677CrossRefPubMedGoogle Scholar
  42. Lemoine MC, Gollotte A, Gianinazzi-Pearson V (1995) Localization of beta (1–3) glucan in walls of the endomycorrhizal fungi Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe and Acaulospora laevis Gerd. & Trappe during colonization of host roots. New Phytol 129:97–105CrossRefGoogle Scholar
  43. Lim CH, Ozkanca R, Flint KP (1996) The effects of osmotic stress on survival and alkaline phosphatase activity of Aeromonas hydrophila. FEMS Microbiol Lett 137:19–24CrossRefGoogle Scholar
  44. Liu J, Blaylock L, Endre G, Cho J, Town CD, VandenBosch KA, Harrison MJ (2003) Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of an arbuscular mycorrhizal symbiosis. Plant Cell 15:2106–2123CrossRefPubMedGoogle Scholar
  45. Manthey K, Krajinski F, Hohnjec N, Firnhaber C, Pühler A, Perlick AM, Küster H (2004) Transcriptome profiling in root nodules and arbuscular mycorrhiza identifies a collection of novel genes induced during Medicago truncatula root endosymbioses. Mol Plant–Microbe Interact 17:1063–1077CrossRefPubMedGoogle Scholar
  46. Martin F, Gianinazzi-Pearson V, Hijri M, Lammers P, Requena N, Sanders IR, Shachar-Hill Y, Shapiro H, Tuskan GA, Young JPW (2008) The long hard road to a completed Glomus intraradices genome. New Phytol 180:747–750CrossRefPubMedGoogle Scholar
  47. Massoumou M, van Tuinen D, Chatagnier O, Arnould C, Brechenmacher L, Sanchez L, Selim S, Gianinazzi S, Gianinazzi-Pearson V (2007) Medicago truncatula gene responses specific to arbuscular mycorrhiza interactions with different species and genera of Glomeromycota. Mycorrhiza 17:223–234CrossRefPubMedGoogle Scholar
  48. Miele R, Borro M, Mangoni ML, Simmaco M, Barra D (2003) A peptidylprolyl cis/trans isomerase from Xenopus laevis skin: cloning, biochemical characterization and putative role in the secretion. Peptides 24:1713–1721CrossRefPubMedGoogle Scholar
  49. Murray A (1995) Cyclin ubiquitination: the destructive end of mitosis. Cell 81:149–152CrossRefPubMedGoogle Scholar
  50. Parniske M (2008) Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nat Rev Microbiol 6:763–775CrossRefPubMedGoogle Scholar
  51. Rivera-Becceril F, Calantzis C, Turnau K, Caussanel J-P, Belimov AA, Gianinazzi S, Strasser RJ, Gianinazzi-Pearson V (2002) Cadmium accumulation and buffering of cadmium-induced stress by arbuscular mycorrhiza in three Pisum sativum L. genotypes. J Exp Bot 53:1177–1185Google Scholar
  52. Sanchez L, Weidmann S, Arnould C, Bernard AR, Gianinazzi S, Gianinazzi-Pearson V (2005) Pseudomonas fluorescens and Glomus mosseae trigger DMI3-dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula. Plant Physiol 139:1065–1077CrossRefPubMedGoogle Scholar
  53. Schnabel E, Journet EP, Carvalho-Niebel F, Duc G, Frugoli J (2005) The Medicago truncatula SUNN gene encodes a CLV1-like leucine-rich repeat receptor kinase the regulates nodule number and root length. Plant Mol Biol 58:809–822CrossRefPubMedGoogle Scholar
  54. Seddas PMA, Arnould C, Tollot M, Arias CM, Gianinazzi-Pearson V (2008) Spatial monitoring of gene activity in extraradical and intraradical developmental stages of arbuscular mycorrhizal fungi by direct fluorescent in situ RT-PCR. Fungal Gen Biol 45:1155–1165CrossRefGoogle Scholar
  55. Seddas PMA, Arias CM, Arnould C, van Tuinen D, Godfroy O, Benhassou HA, Gouzy J, Morandi M, Dessaint F, Gianinazzi-Pearson V (2009) Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions. Mol Plant–Microbe Interact 22:341–351CrossRefPubMedGoogle Scholar
  56. Strittmatter G, Gheysen G, Gianinazzi-Pearson V, Hahn K, Niebel A, Rohde W, Tacke E (1996) Infections with various types of organisms stimulate transcription from a short promoter fragment of the potato gst1 gene. Mol Plant–Microbe Interact 9:68–73PubMedGoogle Scholar
  57. Tisserant B, Gianinazzi-Pearson V, Gianinazzi S, Gollotte A (1993) In planta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections. Mycol Res 97:245–250CrossRefGoogle Scholar
  58. Trouvelot A, Kough J, Gianinazzi-Pearson V (1986) Mesure du taux de mycorhization VA d’un système radiculaire. Recherche de méthodes d’estimation ayant une signification fonctionnelle. In: Gianinazzi-Pearson V, Gianinazzi S (eds) Mycorrhizae: physiology and genetics. INRA, Paris, pp 217–221Google Scholar
  59. Vieheilig H, Coughlan AP, Wyss U, Piche Y (1998) Ink and vinegar, a simple staining technique for arbuscular–mycorrhizal fungi. App Environ Microbiol 64:5004–5007Google Scholar
  60. Weidmann S, Sanchez L, Descombin J, Chatagnier O, Gianinazzi S, Gianinazzi-Pearson V (2004) Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula. Mol Plant–Microbe Interact 17:1385–1393CrossRefPubMedGoogle Scholar
  61. Woolhouse HW (1975) Membrane structure and transport problems considered in relation to phosphorus and carbohydrate movements and the regulation of endotrophic associations. In: Sanders FE, Mosse B, Tinker PB (eds) Endomycorrhizas. Academic Press, London, pp 209–239Google Scholar
  62. Wulf A, Manthey K, Doll J, Perlick AM, Linke B, Bekel T, Meyer F, Franken P, Kuster H, Krajinski F (2003) Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula. Mol Plant–Microbe Interact 16:306–314CrossRefPubMedGoogle Scholar
  63. Zhu H, Choi H-Y, Cook DR, Shoemaker RC (2005) Bridging model and crop legume crops through comparative genomics. Plant Physiol 137:1189–1196CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Elena Kuznetsova
    • 1
    • 2
  • Pascale M. A. Seddas-Dozolme
    • 1
  • Christine Arnould
    • 1
  • Marie Tollot
    • 1
  • Diederik van Tuinen
    • 1
  • Alexey Borisov
    • 2
  • Silvio Gianinazzi
    • 1
  • Vivienne Gianinazzi-Pearson
    • 1
    Email author
  1. 1.UMR 1088 INRA/5184 CNRS/Université de Bourgogne Plante–Microbe–Environnement, INRA-CMSEDijon CedexFrance
  2. 2.Laboratory of Genetics of Plant Microbe Interactions, Department of BiotechnologyAll-Russia Research Institute for Agricultural MicrobiologyPushkin 8Russia

Personalised recommendations