Skip to main content
SpringerLink
Log in

Alfalfa (Medicago sativa L.) resistance to the root-lesion nematode, Pratylenchus penetrans: defense-response gene mRNA and isoflavonoid phytoalexin levels in roots

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Alfalfa (Medicago sativa) varieties with antibiosis-based resistance to the root-lesion nematode (Pratylenchus penetrans), a migratory endoparasite of many crops, have been developed by recurrent selection. Individual plants from these varieties that support significantly lower nematode reproduction were identified for molecular and biochemical characterization of defense responses. Before nematode infection, RNA blot analysis revealed 1.3–1.8-fold higher phenylpropanoid pathway mRNA levels in roots of three resistant plants as compared to three susceptible alfalfa plants. The mRNAs encoded the first enzyme in the pathway (phenylalanine ammonia-lyase), the first in the pathway branch for flavonoid biosynthesis (chalcone synthase), a key enzyme in medicarpin biosynthesis (isoflavone reductase) and a key enzyme in the pathway branch for biosynthesis of lignin cell wall precursors (caffeic acid O-methyltransferase). After nematode infection, the mRNAs declined over 48 h in resistant roots but rose in susceptible plants during the first 12 h after-infection and then declined. Acidic β-1,3-glucanase mRNA levels were initially similar in both root types but accumulated more rapidly in resistant than in susceptible roots after nematode infection. Levels of a class I chitinase mRNA were similar in both root types. Histone H3.2 mRNA levels, initially 1.3-fold higher in resistant roots, declined over 6–12 h to levels found in susceptible roots and remained stable in both root types thereafter. Defense-response gene transcripts in roots of nematode-resistant and susceptible alfalfa plants thus differed both constitutively and in inductive responses to nematode infection. HPLC analysis of isoflavonoid-derived metabolites of the phenylpropanoid pathway revealed similar total constitutive levels, but varying relative proportions and types, in roots of the resistant and susceptible plants. Nematode infection had no effect on isoflavonoid levels. Constitutive levels of the phytoalexin medicarpin were highest in roots of the two most resistant plants. Medicarpin inhibited motility of P. penetrans in vitro.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Barnes DK, Thies JA, Rabas DL, Nelson DL, Smith DM: Registration of two alfalfa germplasms with field resistance to the root lesion nematode. Crop Sci 30: 751–752 (1990).

    Google Scholar 

  2. Basigalup DH: Resistance to Pratylenchus penetrans in alfalfa: mode of inheritance and effects on biological nitrogen fixation. M.S. thesis, Dept. of Agronomy, University of Minnesota (1990).

  3. Bird AF, Bird J: The Structure of Nematodes, 2nd ed., pp. 44–74. Academic Press, San Diego, CA (1991).

    Google Scholar 

  4. Blount JW, Dixon RA, Paiva NL: Stress responses in alfalfa (Medicago sativa L.) XVI. Antifungal activity of medicarpin and its biosynthetic precursors; implications for the genetic manipulation of stress metabolites. Physiol Mol Plant Path 141: 333–349 (1992).

    Google Scholar 

  5. Brosch G, Ransom R, Lechner T, Walton JD, Loidl P: Inhibition of maize deacetylases by HC toxin, the host-selective toxin of Cochliobolus carbonum. Plant Cell 7: 1941–1950 (1995).

    Google Scholar 

  6. Brueske CH: Phenylalanine ammonia lyase activity in tomato roots infected and resistant to the root-knot nematode, Meloidogyne incognita. Physiol Plant Path 16: 409–414 (1980).

    Google Scholar 

  7. Cai, D, Kleine M, Kifle S, Harloff H-J, Sandal NN, Marcker KA, Klein-Lankhorst RM, Salentijn EMJ, Lange W, Stiekma WJ, Wyss U, Grundler FMW, Jung C: Positional cloning of a gene for nematode resistance in sugar beet. Science 275: 832–834 (1997).

    Google Scholar 

  8. Chang LM: The repellent effect of necrotic tissues on the nematode Pratylenchus penetrans (Cobb, 1917), Filipjev and Schurmans Stekhoven, 1941. M.S. thesis, University of Massachusetts (1969).

  9. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium isothyiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159 (1987).

    Google Scholar 

  10. Davis KR, Darvill AG, Albersheim P: Several biotic and abiotic elicitors act synergistically in the induction of phytoalexin accumulation in soybean. Plant Mol Biol 6: 23–32 (1986).

    Google Scholar 

  11. Ebel J, Cosio EG: Elicitors of plant defense responses. Int Rev Cytol 148: 1–31 (1994).

    Google Scholar 

  12. Edens RM, Anand SC, Bolla RI: Enzymes of phenylpropanoid pathway in soybean infected with Meloidogyne incognita or Heterodera glycines. J Nematol 27: 292–303 (1995).

    Google Scholar 

  13. Edwards R, Mizen KA, Cook R: Isoflavonoid conjugate accumulation in the roots of lucerne (Medicago sativa) seedlings following infection by the stem nematode (Ditylenchus dispaci). Nematologica 41: 51–66 (1995).

    Google Scholar 

  14. Engler-Blum G, Meier M, Frank J, Muller GA: Reduction of background problems in nonradioactive northern and southern blot analyses enables higher sensitivity than 32P-based hybridizations. Anal Biochem 210: 235–244 (1993).

    Google Scholar 

  15. Forrest JMS, Robertson WM: Characterization and localization of saccharides on the head region of four populations of the potato cyst nematode Globodera rostochiensis and G. pallida. J Nematol 18: 23–26 (1986).

    Google Scholar 

  16. Gamborg OL, Miller RA, Ojima K: Nutritional requirements of suspension cultures of soybean root cells. Exp Cell Res 50: 151–158 (1968).

    Google Scholar 

  17. Goddijn OJM, Lindsey K, van der Lee FM, Klap JC, Sijmons PC: Differential gene expression in nematode-induced feeding structures of transgenic plants harbouring promoter-gusA fusion constructs. Plant J 4: 863–873 (1993).

    Google Scholar 

  18. Gowri G, Paiva NL, Dixon RA: Stress responses in alfalfa (Medicago sativa L.) 12. Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants. Plant Mol Biol 17: 415–429 (1991).

    Google Scholar 

  19. Gowri G, Bugos RC, Campbell WH, Maxwell CA, Dixon RA: Stress responses in alfalfa (Medicago sativa L.) X. Molecular cloning and expression of S-adenosyl-L-Methionine:caffeic acid 3-O-methyltransferase, a key enzyme of lignin biosynthesis. Plant Physiol 97: 7–14 (1991).

    Google Scholar 

  20. Gurr SJ, McPherson MJ, Scollan C, Atkinson HJ, Bowles DJ: Gene expression in nematode-infected plant roots. Mol Gen Genet 226: 361–366 (1991).

    Google Scholar 

  21. Huang JS, Barker KR: Glyceollin I in soybean-cyst nematode interactions. Plant Physiol 96: 1302–1307 (1991).

    Google Scholar 

  22. Joosten MHAJ, de Wit PJGM: Identification of several pathogenesis-related proteins in tomato leaves inoculated with Cladosporium fulvum (syn. Fulvia fulva) as 1,3-_-glucanases and chitinases. Plant Physiol 89: 945–951 (1989).

    Google Scholar 

  23. Junghans H, Dalkin K, Dixon RA: Stress responses in alfalfa (Medicago sativa L.) 15. Characterization and expression patterns of members of a subset of the chalcone synthase multigene family. Plant Mol Biol 22: 239–253 (1993).

    Google Scholar 

  24. Kaplan DT, Keen NT: Mechanisms conferring plant incompatibility to nematodes. Revue Nematol 3: 123–134 (1980).

    Google Scholar 

  25. Kaplan DT, Keen NT, Thomason IJ: Association of glyceollin with the incompatible response of soybean roots to Meloidogyne incognita. Physiol Plant Pathol 16: 309–318 (1980).

    Google Scholar 

  26. Kaplan DT, Keen NT, Thomason IJ: Studies on the mode of action of glyceollin in soybean incompatibility to the root knot nematode, Meloidogyne incognita. Physiol Plant Path 16: 319–325 (1980).

    Google Scholar 

  27. Kapros T, Bogre L, Nemeth K, Bako L, Gyorgyey J, Wu SC, Dudits D: Differential expression of histone H3 variants during cell cycle and somatic embryogenesis. Plant Physiol 98: 621–625 (1991).

    Google Scholar 

  28. Kessmann H, Edwards R, Geno PW, Dixon RA: Stress responses in alfalfa (Medicago sativa L) V. Constitutive and elicitor-induced accumulation of isoflavonoid conjugates in cell suspension cultures. Plant Physiol 94: 227–232 (1990).

    Google Scholar 

  29. Lambert K, Williamson V: cDNA library construction using PCR. Soc Nematol Mol Biol Newsl 4: 10–11 (1992).

    Google Scholar 

  30. Leath KT, Erwin DC, Griffin GD: Diseases and nematodes. In: Hansen AA, Barnes DK, Hill Jr RR (eds) Alfalfa and Alfalfa Improvement. American Society of Agronomy, Madison, WI,. Monograph 29, pp. 621–670 (1988).

    Google Scholar 

  31. Maher EA, Lamb CJ, Dixon RA: Stress responses in alfalfa (Medicago sativa L.) XVII. Identification of multiple hydrolases and molecular characterization of an acidic glucanase. Physiol Mol Plant Path 43: 329–342 (1993).

    Google Scholar 

  32. Mauch F, Mauch-Mani B, Boller T: Antifungal hydrolases in pea tissue II. Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. Plant Physiol 88: 936–942 (1988).

    Google Scholar 

  33. Miller KJ, Hadley JA, Gustine FL: Cyclic β–1,6–1,3-glucans of Bradyrhizobium japonicum USDA 110 elicit isoflavonoid production in the soybean (Glycine max) host. Plant Physiol 104: 917–923 (1994).

    Google Scholar 

  34. Mittler R, Lam E: Sacrifice in the face of foes: Pathogeninduced programmed cell death in plants. Trends Microbiol 10: 10–15 (1996).

    Google Scholar 

  35. Munch S: Nonradioactive northern blots in 1.5 days and with substantially increased sensitivity through ‘alkaline blotting’. Biochemica 11: 29–31 (1994).

    Google Scholar 

  36. Niebel A, De Almeida Engler J, Tire C, Engler G, Van Montagu M, Gheysen G: Induction patterns of an extensin gene in tobacco upon nematode infection. Plant Cell 5: 1697–1710 (1993).

    Google Scholar 

  37. Niebel A, Gheysen G, Van Montagu M: Plant-cyst nematode and plant-root-knot nematode interactions. Parasitol Today 10: 424–430 (1994).

    Google Scholar 

  38. O'Neill NR: Defense expression in protected tissues of Medicago sativa is enhanced during compatible interactions with Colletotrichum trifolii. Phytopathology 86: 1045–1050 (1996).

    Google Scholar 

  39. Opperman CH, Taylor CG, Conkling MA: Root-knot nematode-directed expression of a plant root-specific gene. Science 263: 221–223 (1994).

    Google Scholar 

  40. Painter RH: The mechanisms of resistance. In Insect Resistance in Crop Plants, pp. 23–83. MacMillan, New York (1951).

    Google Scholar 

  41. Paiva NL, Edwards R, Sun Y, Hrazdina G, Dixon RA: Stress responses in alfalfa (Medicago sativa L.). 11. Molecular cloning and expression of isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis. Plant Mol Biol 17: 653–667 (1991).

    Google Scholar 

  42. Potenza CL, Thomas SH, Higgins EA, Sengupta-Gopalan C: Early root response to Meloidogyne incognita in resistant and susceptible alfalfa cultivars. J Nematol 28: 475–484 (1996).

    Google Scholar 

  43. Rahimi S, Perry RN, Wright DJ: Identification of pathogenesis-related proteins induced in leaves of potato plants infected with potato cyst nematodes, Globodera species. Physiol Mol Plant Path 49: 49–59 (1996).

    Google Scholar 

  44. Rich JR, Keen NT, Thomason: Association of coumestans with the hypersensitivity of lima bean roots to Pratylenchus scribneri. Physiol Plant Path 10: 105–116 (1977).

    Google Scholar 

  45. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, NY (1989).

    Google Scholar 

  46. Sela-Buurlage MB, Ponstein As, Bres-Vloemans SA, Melchers LS, van den Elzen PJM, Cornelissen BJC: Only specific tobacco (Nicotiana tabacum) chitinases and β-1,3-glucanases exhibit antifungal activity. Plant Physiol 101: 857–863 (1993).

    Google Scholar 

  47. Sijmons PC: Plant-nematode interactions. Plant Mol Biol 23: 17–931 (1993).

    Google Scholar 

  48. Smith CJ: Accumulation of phytoalexins: defense mechanism and stimulus response. New Phytol 132: 1–45 (1996).

    Google Scholar 

  49. Spanakis E: Problems related to the interpretation of autoradiographic data on gene expression using common constitutive transcripts as controls. Nucl Acids Res 21: 3809–3819 (1993).

    Google Scholar 

  50. Spiegel Y, Cohn E, Spiegel S: Characterization of sialyl and galactosyl residues on the body wall of different plant parasitic nematodes. J Nematol 14: 33–39 (1982).

    Google Scholar 

  51. Stevenson PC, Turner HC, Haware MP: Phytoalexin accumulation in the roots of chickpea (Cicer arietinum L.) seedlings associated with resistance of fusarium wilt (Fusarium oxysporum f.sp. ciceri). Physiol Mol Plant Path 50: 167–178 (1997).

    Google Scholar 

  52. Thies JA, Basigalup D, Barnes DK: Inheritance of resistance to Pratylenchus penetrans in alfalfa. J Nematol 26: 452–459 (1994).

    Google Scholar 

  53. Thies JA, Petersen AD, Barnes DK: Host suitability of forage grasses and legumes for root-lesion nematode Pratylenchus penetrans. Crop Sci 35: 1647–1651 (1995).

    Google Scholar 

  54. Tiller SA, Parry AD Edwards R: Changes in the accumulation of flavonoid and isoflavonoid conjugates associated with plant age and nodulation in alfalfa (Medicago sativa). Physiol Plant 91: 27–36 (1994).

    Google Scholar 

  55. Townshend JL, Stobbs L, Carter R: Ultrastructural pathology of cells affected by Praylenchus penetrans in alfalfa roots. J Nematol 21: 530–539 (1989).

    Google Scholar 

  56. Townshend JL, Stobbs L: Histopathology and histochemistry of lesions caused by Pratylenchus penetrans in roots of forage legumes. Can J Plant Path 3: 123–186 (1981).

    Google Scholar 

  57. Trudgill DL: Resistance to and tolerance of plant parasitic nematodes. Annu Rev Phytopath 29: 167–192 (1991).

    Google Scholar 

  58. Van der Eycken W, Engler J, Inzé D, Van Montagu M, Gheysen G: A molecular study of root-knot nematode-induced feeding sites. Plant J 9: 45–54 (1996).

    Google Scholar 

  59. Wu SC, Grorgyey J, Dudits D: Polyadenylated H3 histone transcripts and H3 histone variants in alfalfa. Nucl Acids Res 17: 3057–3063 (1989).

    Google Scholar 

  60. Wyss U: Ectoparasitic root nematodes: Feeding behavior and plant cell responses. In: Zuckerman BM, Rohde RA (eds) Plant Parasitic Nematodes, pp. 211–220. Academic Press, New York (1981).

    Google Scholar 

  61. Zacheo G, Bleve-Zacheo T, Lamberti F: Role of peroxide and superoxide dismutase in resistant and susceptible tomato cultivars infested by Meloidogyne incognita. Nematol Mediterr 10: 75–80 (1982).

    Google Scholar 

  62. Zunke, U: Observations on the invasion and endoparasitic behavior of the root lesion nematode Pratylenchus penetrans. J Nematol 22: 309–320 (1990).

    Google Scholar 

Download references

Author information

Author notes

  1. Deborah A. Samac

    Present address: Department of Plant Pathology, University of Minnesota, 495 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN, 55108, USA

Authors and Affiliations

  1. USDA-ARS-Plant Science Research Unit, USA

    Deborah A. Samac

  2. USDA-ARS-Soybean and Alfalfa Research Lab, Beltsville, MD, 20705, USA

    Gerald D. Baldridge & Nichole R. O'Neill

Authors
  1. Gerald D. Baldridge
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nichole R. O'Neill
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Deborah A. Samac
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Baldridge, G.D., O'Neill, N.R. & Samac, D.A. Alfalfa (Medicago sativa L.) resistance to the root-lesion nematode, Pratylenchus penetrans: defense-response gene mRNA and isoflavonoid phytoalexin levels in roots. Plant Mol Biol 38, 999–1010 (1998). https://doi.org/10.1023/A:1006182908528

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1006182908528

Search

Navigation