Skip to main content
SpringerLink
Log in

Application of differential display RT-PCR to the analysis of gene expression in a plant-fungus interaction

  • Research Article
  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Establishment of a plant-pathogen interaction involves differential gene expression in both organisms. In order to isolate Botrytis cinerea genes whose expression is induced during its interaction with tomato, a comparative analysis of the expression pattern of the fungus in planta with its expression pattern during in vitro culture was performed by differential display of mRNA (DDRT-PCR). Discrimination of fungal genes induced in planta from plant defense genes induced in response to the pathogen was attempted by including in this comparative analysis the expression patterns of healthy tomato leaves and of tomato leaves infected with two different pathogens, either Phytophthora infestans or tobacco necrosis virus (TNV). Using a limited set of primer combinations, three B. cinerea cDNA fragments, ddB-2, ddB-5 and ddB-47, were isolated representing fungal genes whose expression is enhanced in planta. Northern blot analysis showed that the transcripts detected with the cDNA clones ddB-2 and ddB-5 accumulated at detectable levels only at late time points during the interaction. The cDNA clone ddB-47 detected two different sizes of transcripts displaying distinct, transient expression patterns during the interaction. Sequence analysis and database searches revealed no significant homology to any known sequence. These results show that the differential display procedure possesses enough sensitivity to be applied to the detection of fungal genes induced during a plant-pathogen interaction. Additionally, four cDNA fragments were isolated representing tomato genes induced in response to the infection caused by B. cinerea, but not by P. infestans.

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. Appleyard VCL, Unkles SE, Legg M, Kinghorn JR: Secondary metabolite production in filamentous fungi displayed. Mol Gen Genet 247: 338–342 (1995).

    Article  PubMed  Google Scholar 

  2. Bauer D, Müller H, Reich J, Riedel H, Ahrenkiel V, Warthoe P, Strauss M: Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR). Nucl Acids Res 21: 4272–4280 (1993).

    PubMed  Google Scholar 

  3. Bertioli DJ, Schlichter UHA, Adams MJ, Burrows PR, Steinbiss H-H, Antoniw JF: An analysis of differential display shows a strong bias towards high copy number mRNAs. Nucl Acids Res 23: 4520–4523 (1995).

    PubMed  Google Scholar 

  4. Bhairi SM, Staples RC, Freve P, Yoder OC: Characterization of an infection-specific gene from the rust fungus Uromyces appendiculatus. Gene 81: 237–243 (1989).

    Article  PubMed  Google Scholar 

  5. Caten CE, Jinks JL: Spontaneous variability of single isolates of Phytophthora infestans I. Cultural variation. Can J Bot 46: 329–347 (1968).

    Google Scholar 

  6. Church GM, Gilbert W: Genomic sequencing. Proc Natl Acad Sci USA 81: 1991–1995 (1984).

    PubMed  Google Scholar 

  7. Faretra F, Antonacci E, Pollastro S: Sexual behaviour and mating system of Botryotinia fuckeliana, teleomorph of Botrytis cinerea. J Gen Microbiol 134: 2543–2550 (1988).

    Google Scholar 

  8. Fermaud M, LeMenn R: Association of Botrytis cinerea with grape berry moth larvae. Phytopathology 79: 651–656 (1989).

    Google Scholar 

  9. Goormachtig S, Valerio-Lepiniec M, Szczyglowsky K, VanMontagu M, Holsters M, deBruijn FJ: Use of differential display to identify novel Sesbania rostrata genes enhanced by Azorhizobium caulinodans infection. Mol Plant-Microbe Interact 8: 816–824 (1995).

    PubMed  Google Scholar 

  10. Hahlbrock K, Scheel D: Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40: 347–369 (1989).

    Article  Google Scholar 

  11. Hwang C-S, Kolattukudy P: Isolation and characterization of genes expressed uniquely during appressorium formation by Colletotrichum gloeosporioides conidia induced by the host surface wax. Mol Gen Genet 247: 282–294 (1995).

    PubMed  Google Scholar 

  12. Jarvis W: Botryotinia and Botrytis Species: Taxonomy and Pathogenicity. Canada Department of Agriculture Monograph 15 (1977).

  13. Johnston DJ, Williamson B: Purification and characterization of four polygalacturonases from Botrytis cinerea. Mycol Res 96: 343–349 (1992).

    Google Scholar 

  14. Lamb CJ, Lawton MA, Dron M, Dixon RA: Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell 56: 215–224 (1989).

    Article  PubMed  Google Scholar 

  15. Liang P, Pardee AB: Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967–971 (1992).

    PubMed  Google Scholar 

  16. Liang P, Zhu W, Zhang X, Guo Z, O'Connell RP, Averboukh L, Wang F, Pardee AB: Differential display using one-base anchored oligo-dT primers. Nucl Acids Res 22: 5763–5764 (1994).

    PubMed  Google Scholar 

  17. Liang P, Pardee AB: Recent advances in differential display. Curr Opin Immunol 7: 274–280 (1995).

    Article  PubMed  Google Scholar 

  18. Logemann J, Schell J, Willmitzer L: Improved method for the isolation of RNA from plant tissues. Anal Biochem 163: 16–20 (1987).

    PubMed  Google Scholar 

  19. Mansfield JW: Mechanisms of resistance to Botrytis. In: Coley-Smith JR, Verhoeff K, Jarvis WR (eds) The Biology of Botrytis, pp 181–218. Academic Press, New York/London (1980).

    Google Scholar 

  20. McClellan WD, Hewitt WB: Early Botrytis rot in grapes: time of infection and latency of Botrytis cinerea Pers. in Vitis vinifera L. Phytopathology 63: 1151–1157 (1973).

    Google Scholar 

  21. Mizutani M, Ward E, Dimaio J, Ohta D, Ryals J, Sato R: Molecular cloning and sequencing of a cDNA encoding mung bean cytochrome P450 (P450C4H) possessing 4-hydroxylase activity. Biochem Biophys Res Commun 190: 875–880 (1993).

    Article  PubMed  Google Scholar 

  22. Möller EM, Bahnweg G, Sandermann H, Geiger HH: A simple and efficient protocol for isolation of high molecular weight DNA filamentous fungi, fruit bodies, and infected plant tissues. Nucl Acids Res 20: 6115–6116 (1992).

    PubMed  Google Scholar 

  23. Movahedi S, Heale JB: The roles of aspartic proteinase and endo-pectin lyase enzymes in the primary stages of infection and pathogenesis of various host tissues by different isolates of Botrytis cinerea Pers ex. Pers. Physiol Mol Plant Path 36: 303–324 (1990).

    Google Scholar 

  24. Pieterse CMJ, Riach MBR, Bleker T, van denBerg-Velthuis GCM, Govers F: Isolation of putative pathogenicity genes of the potato late blight fungus Phytophthora infestans by differential hybridization of a genomic library. Physiol Mol Plant Path 43: 69–79 (1993).

    Article  Google Scholar 

  25. Reignault P, Mercier M, Bompeix G, Boccara M: Pectin methylesterase from Botrytis cinerea physiological, biochemical and immunochemical studies. Microbiology 140: 3249–3255 (1994).

    Google Scholar 

  26. Salinas J, Glandorf DCM, Picavet FD, Verhoeff K: Effects of temperature, relative humidity and age of conidia in the incidence of spotting on gerbera flowers caused by Botrytis cinerea. Neth J Plant Path 95: 51–64 (1989).

    Google Scholar 

  27. Salinas J: Function of cutinolytic enzymes in the infection of gerbera flowers by Botrytis cinerea. Ph.D. thesis, University of Utrecht, Netherlands (1992).

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

    Google Scholar 

  29. Sargent TD: Isolation of differentially expressed genes. In: Berger SL, Kimmel AR (eds). Methods in Enzymology, Volume 152. Guide to Molecular Cloning Techniques, pp. 423–432. Academic Press, San Diego (1987).

    Google Scholar 

  30. Sharma YK, Davis KR: Isolation of a novel Arabidopsis ozone-induced cDNA by differential display. Plant Mol Biol 29: 91–98 (1995).

    PubMed  Google Scholar 

  31. Talbot NJ, Ebbole DJ, Hamer JE: Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5: 1575–1590 (1993).

    Article  PubMed  Google Scholar 

  32. Teutsch HG, Hasenfratz MP, Lesot A, Stoltz C, Garnier J-M, Jeltsch JM, Durst F, Werck-Reichhart D: Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway. Proc Natl Acad Sci USA 90: 4102–4106 (1993).

    PubMed  Google Scholar 

  33. van derKnaap E, Kende H: Identification of a gibberellin-induced gene in deepwater rice using differential display of mRNA. Plant Mol Biol 28: 589–592 (1995).

    PubMed  Google Scholar 

  34. Wilkinson JQ, Lanahan MB, Conner TW, Klee HJ: Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display. Plant Mol Biol 27: 1097–1108 (1995).

    PubMed  Google Scholar 

  35. Wilson AR: Some observations on the infections of tomato stems by Botrytis cinerea. Ann Appl Biol 51: 171 (1963).

    Google Scholar 

  36. Zimmermann JW, Schultz RM: Analysis of gene expression in the preimplantation mouse embryo: use of the mRNA differential display. Proc Natl Acad Sci USA 91: 5456–5460 (1994).

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Phytopathology, Wageningen Agricultural University, P.O. Box 8025, 6700 EE, Wageningen, Netherlands

    Ernesto P. Benito, Theo Prins & Jan A. L. van Kan

Authors
  1. Ernesto P. Benito
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Theo Prins
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jan A. L. van Kan
    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

Benito, E.P., Prins, T. & van Kan, J.A.L. Application of differential display RT-PCR to the analysis of gene expression in a plant-fungus interaction. Plant Mol Biol 32, 947–957 (1996). https://doi.org/10.1007/BF00020491

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00020491

Key words

Search

Navigation