Plant Molecular Biology

, Volume 15, Issue 6, pp 797–808 | Cite as

Evidence for a third structural class of β-1,3-glucanase in tobacco

  • George Payne
  • Eric Ward
  • Thomas Gaffney
  • Patricia Ahl Goy
  • Mary Moyer
  • Andrew Harper
  • Frederick MeinsJr
  • John Ryals


Glucan endo-1,3-β-glucosidases (β-1,3-glucanases) have been implicated in several developmental processes and they may also play a direct role in the plant's defense against fungal pathogens. In an effort to characterize the glucanase gene family, complementary DNA clones encoding an acidic form of β-1,3-glucanase have been isolated from tobacco. The cDNA was expressed in E. coli and shown to encode a β-1,3-glucanase activity. The protein sequence encoded by the cDNA was found to match the partial protein sequence of PR-35, a previously characterized β-1,3-glucanase [29]. The protein encoded by the cDNA was purified from the extracellular fluid of TMV-infected tobacco leaves and found by immunological methods to correspond to glucanase PR-Q' [10]. From a detailed analysis of the cDNA it is clear that this glucanase represents a third structural class of enzyme which differs substantially from both the basic, vacuolar glucanase and the acidic, extracellular forms (PR-2, PR-N and PR-O). It has previously been demonstrated that the basic form of β-1,3-glucanase is synthesized as a pre-pro-enzyme and upon maturation the 21 amino acid signal peptide and a 22 amino acid carboxy-terminal peptide are removed. This processing event has been proposed to be involved with the vacuolar localization of the enzyme. By comparing the deduced protein structure of PR-Q' to that of the basic form it is evident that this extracellular enzyme is missing the carboxy-terminal 22 amino acids. The role of a conserved phenylalanine-glycine dipeptide in the processing of glucanases and other pathogenesis-related proteins from tobacco is discussed.

Key words

glucanase cDNA cloning Nicotiana tabacum pathogenesis-related proteins plant defense genes 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Amann E, Brosius J: ‘ATG’ vectors for regulated highlevel expression of cloned genes in Escherichia coli. Gene 40: 183–190 (1985).CrossRefPubMedGoogle Scholar
  2. 2.
    Ausubel F, Brent R, Kingston R, Moore D, Seidman J, Smith J, Struhl K: Current Protocols in Molecular Biology. J. Wiley and Sons, New York (1987).Google Scholar
  3. 3.
    Boller T: Ethylene and the regulation of antifungal hydrolases in plants. Oxf Surv Plant Mol Cell Biol 5: 145–174 (1988).Google Scholar
  4. 4.
    Carr JP, Klessig DF: The pathogenesis-related proteins of plants. In: Setlow JK (ed) Genetic Engineering, vol. 11. Plenum Press, New York (1989).Google Scholar
  5. 5.
    Church G, Gilbert W: Genomic sequencing. Proc Natl Acad Sci (USA) 81: 1991–1995 (1984).Google Scholar
  6. 6.
    Denecke J, Botterman J, Deblaere R: Protein secretion in plant cells can occur via a default pathway. Plant Cell 2: 51–59 (1990).CrossRefPubMedGoogle Scholar
  7. 7.
    Dore C, Voelker TA, Herman EM, Chrispeels MJ: 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).CrossRefPubMedGoogle Scholar
  8. 8.
    Felix G, Meins F: Developmental and hormonal regulation of β-1,3-glucanase in tobacco. Planta 167: 206–211 (1986).Google Scholar
  9. 9.
    Fincher GB, Stone BA: Metabolism of noncellulosic polysaccharides. Enycl Plant Physol New Series 13B: 68–132 (1981).Google Scholar
  10. 10.
    Fritig B, Kauffmann S, Rouster J, Dumas B, Geoffroy P, Kopp M, Legrand M: Defense proteins, glycanohydrolases and oligosaccharide signals in plant-virus interactions. In: Fraser RSS (ed) Recognition and Response in Plant-Virus Interactions, pp 325–394. Springer, Berlin (1990).Google Scholar
  11. 11.
    hattoni M, Sakaki Y: Dideoxy sequencing method using denatured plasmid templates. Anal Biochem 152: 232–238 (1986).PubMedGoogle Scholar
  12. 12.
    Kaufmann S, Legrand M, Geoffroy P, Fritig B: Biological function of ‘pathogenesis-related” proteins: four PR proteins of tobacco have β-1,3-glucanase activity. EMBO J 6: 3209–3212 (1987).Google Scholar
  13. 13.
    Keefe D, Hinz U, Meins F: The effect of ethylene on the cell-type-specific and intracellular localization of β-1,3-glucanase and chitinase in tobacco leaves. Planta (1990) In Press.Google Scholar
  14. 14.
    Keen NT, Yoshikawa M: β-1,3-Endoglucanase from soybean releases elicitor-active carbohydrates from fungus cell walls. Plant Physiol 71: 460–465 (1983).Google Scholar
  15. 15.
    Kombrink E, Schröder M, Hahlbrock K: Several ‘pathogenesis-related’ proteins in potato are 1,3-β-glucanases and chitinases. Proc Natl Acad Sci USA 85: 782–786 (1988).Google Scholar
  16. 16.
    Lagrimini M, Burkhart W, Moyer M, Rothstein S: Molecular cloning of complementary DNA encoding the lignin-forming peroxidase from tobacco: Molecular analysis and tissue-specific expression. Proc Natl Acad Sci USA 84: 7542–7546 (1987).Google Scholar
  17. 17.
    Mauch F, Mauch-Mani B, Boller T: Antifungal hydrolases II. Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. Plant Physiol 88: 936–942 (1988).Google Scholar
  18. 18.
    Mauch F, Staehelin LA: Functional implications of the subcellular localization of ethylene-induced chitinase and β-1,3-glucanase in bean leaves. Plant Cell 1: 447–457 (1989).CrossRefPubMedGoogle Scholar
  19. 19.
    Meins F, Ahl P: 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).CrossRefGoogle Scholar
  20. 20.
    Mohnen D, Shinshi H, Felix G, Meins F: Hormonal regulation of β-1,3-glucanase messenger RNA levels in cultured tobacco tissues. EMBO J 4: 1631–1635 (1985).Google Scholar
  21. 21.
    Payne G, Parks TD, Burkhart W, Dincher S, Ahl P, metraux JP, Ryals J: Isolation of the genomic clone for pathogenesis-related protein 1a from Nicotiana tabacum cv. Xanthi-nc. Plant Mol Biol 11: 89–94 (1988).Google Scholar
  22. 22.
    Payne G, Ahl P, Moyer M, Harper A, Beck J, Meins F, Ryals J: 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).PubMedGoogle Scholar
  23. 23.
    Pfitzner UM, Goodman HM: Isolation and characterization of cDNA clones encoding pathogenesis-related proteins from tobacco mosaic virus infected tobacco plants. Nucleic Acids Res 15: 4449–4465 (1987).PubMedGoogle Scholar
  24. 24.
    Reed KC, Mann DA: Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13: 7207–7221 (1985).PubMedGoogle Scholar
  25. 25.
    Shinshi H, Mohnen D, Meins F: 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
  26. 26.
    Shinshi H, Wenzler H, Neuhaus J-M, Felix G, Hofsteenge J, Meins F: 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
  27. 27.
    Shinshi H, Neuhaus J-M, Ryals J, Meins F: 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).PubMedGoogle Scholar
  28. 28.
    Takeuchi Y, Yoshikawa M, Takeba G, Kunisuke T, Shibata D, Horino O: Molecular cloning and ethylene induction of mRNA encoding a phytoalexin elicitorreleasing factor β-1,3-endoglucanase, in soybean. Plant Physiol 93: 673–682 (1990).Google Scholar
  29. 29.
    Van den Bulcke M, Bauw G, Castresana C, Van Montagu M: Characterization of vacuolar and extracellular β(1,3)-glucanases of tobacco: Evidence for a strictly compartmentalized plant defense system. Proc Natl Acad Sci USA 86: 2673–2677 (1989).Google Scholar
  30. 30.
    Van Loon LC: Pathogenesis-related proteins. Plant Mol Biol 4: 111–116 (1985).Google Scholar
  31. 31.
    Vogeli-Lange R, Hansen-Gehri A, Boller T, Meins F: Induction of the defense-related glucanohydrolases, β-1,3-glucanase and chitinase, by tobacco mosaic virus infection of tobacco leaves. Plant Sci 4: 171–176 (1988).CrossRefGoogle Scholar
  32. 32.
    Wessels JG, Sietsma JH: Fungal cell walls: a survey. In: Tanner W, Loewus FA (eds) Encylopedia of Plant Physiology, N.S., vol. 13B, Plant carbohydrates II, pp. 352–394. Springer, Berlin.Google Scholar
  33. 33.
    Yanisch-Perron C, Vieira J, Messing J: Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103–113.Google Scholar

Copyright information

© Kluwer Academic Publishers 1990

Authors and Affiliations

  • George Payne
    • 1
  • Eric Ward
    • 1
  • Thomas Gaffney
    • 1
  • Patricia Ahl Goy
    • 2
  • Mary Moyer
    • 1
  • Andrew Harper
    • 1
  • Frederick MeinsJr
    • 3
  • John Ryals
    • 1
  1. 1.CIBA-GEIGY Agricultural Biotechnology Research UnitResearch Triangle ParkUSA
  2. 2.Agricultural DivisionCIBA-GEIGY LimitedBaselSwitzerland
  3. 3.Friedrich Miescher-InstitutBaselSwitzerland

Personalised recommendations