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Plant Molecular Biology

, Volume 16, Issue 1, pp 141–151 | Cite as

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

  • Jean-Marc Neuhaus
  • Patricia Ahl-Goy
  • Ursula Hinz
  • Susan Flores
  • Frederick MeinsJr.
Article

Abstract

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.

Key words

plant defense genes protein processing β-1,3-glucanase Frog Eye disease fungal pathogens gene-silencing 

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References

  1. 1.
    BenfeyPN, ChuaN-H: Regulated genes in transgenic plants. Science 244: 174–181 (1989).Google Scholar
  2. 2.
    BollerT: Ethylene and the regulation of antifungal hydrolases in plants. Oxford Surveys Plant Molec Cell Biol 5: 145–174 (1988).Google Scholar
  3. 3.
    BollerT, KendeH: Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol 63: 1123–1132 (1979).Google Scholar
  4. 4.
    BollerT, VögeliU: Vacuolar localization of ethylene-induced chitinase in bean leaves. Plant Physiol 74: 442–444 (1984).Google Scholar
  5. 5.
    CarrJP, KlessigDF: The pathogenesis-related proteins of plants. Genet Engineering 11: 65–109 (1989).Google Scholar
  6. 6.
    DorelC, VoelkerTA, HermanEM, ChrispeelsMJ: Transport of proteins to the plant vacuole is not by bulk flow through the secretory system and requires positive sorting information. J Cell Biol 108: 327–337 (1989).Google Scholar
  7. 7.
    FelixG, MeinsFJr: Purification, immunoassay and characterization of an abundant, cytokinin-regulated polypeptide in cultured tobacco tissues. Evidence the protein is a β-1,3-glucanase. Planta 164: 423–428 (1985).Google Scholar
  8. 8.
    FelixG, MeinsFJr: Developmental and hormonal regulation of β-1,3-glucanase in tobacco. Planta 167: 206–211 (1986).Google Scholar
  9. 9.
    GasparT, PenelC, ThorpeT, GreppinH: Peroxidases 1970–1980. University Centre Botanique, Geneva (1982).Google Scholar
  10. 10.
    HoekemaA, HirschPR, HooykaasPJJ, SchilperoortRA: A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180 (1983).Google Scholar
  11. 11.
    HorschRB, FryJE, HoffmannNL, EichholtzD, RogersSG, FraleyRT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).Google Scholar
  12. 12.
    JeffersonRA, KavanaghTA, BevanMW: Gus fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).Google Scholar
  13. 13.
    JonesJDG, DeanC, GidoniD, GilbertD, Bond-NutterD, LeeR, BedbrookJ, DunsmuirP: Expression of bacterial chitinase protein in tobacco leaves using two photosynthetic gene promoters. Mol Gen Genet 212: 536–542 (1988).Google Scholar
  14. 14.
    KeefeD, HinzU, MeinsFJr: The effect of ethylene on the cell-type specific and intracellular localization of β-1,3-glucanase and chitinase in tobacco leaves. Planta 182: 43–51 (1990).Google Scholar
  15. 15.
    LaurellCB, McKayEJ: Electroimmunoassay. Methods Enzymol 73: 339–369 (1981).Google Scholar
  16. 16.
    LinsmaierEM, SkoogF: Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: 100–127 (1965).Google Scholar
  17. 17.
    LogemannJ, SchellJ, WillmitzerL: Improved method for the isolation of RNA from plant tissues. Analyt Biochem 163: 16–20 (1987).Google Scholar
  18. 18.
    LucasGB: Diseases of Tobacco. Biological Consulting Associates, Raleigh (1975).Google Scholar
  19. 19.
    ManiatisT, FritschEF, SambrookJ: Molecular Cloning. A laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1982).Google Scholar
  20. 20.
    MauchF, Mauch-ManiB, BollerT: 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
  21. 21.
    MauchF, StaehelinLA: Functional implications of the subcellular localization of ethylene-induced chitinase and β-1,3-glucanase in bean leaves. Plant Cell 1: 447–457 (1989).Google Scholar
  22. 22.
    MeinsFJr, AhlP: Induction of chitinase and β-1,3-glucanase in tobacco plants infected with Pseudomonas tabaci and Phytophthora parasitica var. nicotianae. Plant Sci 61: 155–161 (1989).Google Scholar
  23. 23.
    MohnenD, ShinshiH, FelixG, MeinsFJr: Hormonal regulation of β-1,3-glucanase messenger RNA levels in cultured tobacco tissues. EMBO J 4: 1631–1635 (1985).Google Scholar
  24. 24.
    MurrayMG, ThompsonWF: Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8: 4321–4325 (1980).Google Scholar
  25. 25.
    NapoliC, LemieuxC, JorgensenR: Introduction of a chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279–289 (1990).Google Scholar
  26. 26.
    ParentJG, AsselingA: Detection of pathogenesis-related proteins (PR or b) and of other proteins in the intercellular fluid of hypersensitive plants infected with tobacco mosaic virus. Can J Bot 62: 564–569 (1984).Google Scholar
  27. 27.
    PayneG, AhlP, MoyerM, HarperA, BechJ, MeinsFJr, RyalsJ: Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco. Proc Natl Acad Sci USA 87: 98–102 (1990).Google Scholar
  28. 28.
    PietrzakM, ShillitoRD, HohnT, PotrykusI: Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucleic Acid Res 14: 5857–5868 (1986).Google Scholar
  29. 29.
    RothsteinSJ, LahnersKN, LotsteinRJ, CarozziNB, JaynesSM, RiceDA: Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation. Gene 53: 153–161 (1987).Google Scholar
  30. 30.
    SchlossP, WalterC, MäderM: Basic peroxidases in isolated vacuoles of Nicotiana tabacum L. Planta 170: 225–229 (1987).Google Scholar
  31. 31.
    SchlumbaumA, MauchF, VögeliU, BollerT: Plant chitinases are potent inhibitors of fungal growth. Nature 324: 365–367 (1986).Google Scholar
  32. 32.
    SchmidhauserTJ, HelinskiDR: Ti regions of broad-host-range plasmid RK-2 involved in replication and stable maintenance of nine species of gram-negative bacteria. J Bact 164: 446–455 (1985).Google Scholar
  33. 33.
    ShinshiH, MohnenD, MeinsFJr: Regulation of a plant pathogenesis-related enzyme: Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin. Proc Natl Acad Sci USA 84: 89–93 (1987).Google Scholar
  34. 34.
    ShinshiH, NeuhausJ-M, RyalsJ, MeinsFJr: Structure of a tobacco endochitinase gene: evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain. Plant Mol Biol 14: 357–368 (1990).Google Scholar
  35. 35.
    ShinshiH, WenzlerH, NeuhausJ-M, FelixG, HofsteengeJ, MeinsFJr: Evidence for N- and C-terminal processing of a plant defense-related enzyme: Primary structure of tobacco prepro-β-1,3-glucanase. Proc Natl Acad Sci USA 85: 5541–5545 (1988).Google Scholar
  36. 36.
    SeigelBZ, GalstonAW: The isoperoxidases of Pisum sativum. Plant Physiol 42: 221–223 (1967).Google Scholar
  37. 37.
    van derKrolAR, MurLA, BeldM, MolJNM, StuitjeAR: Flavonoid genes in Petunia: Addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2: 291–299 (1990).Google Scholar
  38. 38.
    VieiraJ, MessingJ: The PUC plasmids. An m-13MP-7 derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: 259–268 (1982).Google Scholar
  39. 39.
    Vögeli-LangeR, Hansen-GehriA, BollerT, MeinsFJr: Induction of the defense-related glucanohydrolases, β-1,3-glucanase and chitinase, by tobacco mosaic virus infection of tobacco leaves. Plant Sci 54: 171–176 (1988).Google Scholar

Copyright information

© Kluwer Academic Publishers 1991

Authors and Affiliations

  • Jean-Marc Neuhaus
    • 1
  • Patricia Ahl-Goy
    • 2
  • Ursula Hinz
    • 1
  • Susan Flores
    • 1
  • Frederick MeinsJr.
    • 1
  1. 1.Friedrich Miescher-InstitutBaselSwitzerland
  2. 2.Ciba-Geigy, AGBaselSwitzerland

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