Abstract
Different isoforms of chitinases and [beta]-1,3-glucanases of tobacco (Nicotiana tabacum cv Samsun NN) were tested for their antifungal activities. The class I, vacuolar chitinase and [beta]-1,3-glucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In additon, we observed that the class I chitinase and [beta]-1,3-glucanase acted synergistically. The class II isoforms of the two hydrolases exhibited no antifungal activity. However, the class II chitinases showed limited growth inhibitory activity in combination with higher amounts of class I [beta]-1,3-glucanase. The class II [beta]-1,3-glucanases showed no inhibitory activity in any combination. In transgenic tobacco plants producing modified forms of either a class I chitinase or a class I [beta]-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their C-terminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and [beta]-1,3-glucanase enzymes. Furthermore, the extracellular washing fluid (EF) from leaves of transgenic plants expressing either of the secreted class I enzymes exhibited antifungal activity on F. solani germlings in vitro comparable to that of the purified vacuolar class I proteins. Mixing EF fractions from these plants revealed synergism in inhibitory activity against F. solani; the mixed fractions exhibited inhibitory activity similar to that of EF from plants expressing both secreted enzymes.
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- Bowles D. J. Defense-related proteins in higher plants. Annu Rev Biochem. 1990;59:873–907. doi: 10.1146/annurev.bi.59.070190.004301. [DOI] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Brederode F. T., Linthorst H. J., Bol J. F. Differential induction of acquired resistance and PR gene expression in tobacco by virus infection, ethephon treatment, UV light and wounding. Plant Mol Biol. 1991 Dec;17(6):1117–1125. doi: 10.1007/BF00028729. [DOI] [PubMed] [Google Scholar]
- Kauffmann S., Legrand M., Geoffroy P., Fritig B. Biological function of ;pathogenesis-related' proteins: four PR proteins of tobacco have 1,3-beta-glucanase activity. EMBO J. 1987 Nov;6(11):3209–3212. doi: 10.1002/j.1460-2075.1987.tb02637.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keen N. T., Yoshikawa M. beta-1,3-Endoglucanase from Soybean Releases Elicitor-Active Carbohydrates from Fungus Cell Walls. Plant Physiol. 1983 Mar;71(3):460–465. doi: 10.1104/pp.71.3.460. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lawton K., Ward E., Payne G., Moyer M., Ryals J. Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco. Plant Mol Biol. 1992 Aug;19(5):735–743. doi: 10.1007/BF00027070. [DOI] [PubMed] [Google Scholar]
- Legrand M., Kauffmann S., Geoffroy P., Fritig B. Biological function of pathogenesis-related proteins: Four tobacco pathogenesis-related proteins are chitinases. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6750–6754. doi: 10.1073/pnas.84.19.6750. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Linthorst H. J., van Loon L. C., van Rossum C. M., Mayer A., Bol J. F., van Roekel J. S., Meulenhoff E. J., Cornelissen B. J. Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco. Mol Plant Microbe Interact. 1990 Jul-Aug;3(4):252–258. doi: 10.1094/mpmi-3-252. [DOI] [PubMed] [Google Scholar]
- Mauch F., Mauch-Mani B., Boller T. Antifungal Hydrolases in Pea Tissue : II. Inhibition of Fungal Growth by Combinations of Chitinase and beta-1,3-Glucanase. Plant Physiol. 1988 Nov;88(3):936–942. doi: 10.1104/pp.88.3.936. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mauch F., Staehelin L. A. Functional Implications of the Subcellular Localization of Ethylene-Induced Chitinase and [beta]-1,3-Glucanase in Bean Leaves. Plant Cell. 1989 Apr;1(4):447–457. doi: 10.1105/tpc.1.4.447. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Molano J., Durán A., Cabib E. A rapid and sensitive assay for chitinase using tritiated chitin. Anal Biochem. 1977 Dec;83(2):648–656. doi: 10.1016/0003-2697(77)90069-0. [DOI] [PubMed] [Google Scholar]
- Neuhaus J. M., Sticher L., Meins F., Jr, Boller T. A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10362–10366. doi: 10.1073/pnas.88.22.10362. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Payne G., Ahl P., Moyer M., Harper A., Beck J., Meins F., Jr, Ryals J. Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco. Proc Natl Acad Sci U S A. 1990 Jan;87(1):98–102. doi: 10.1073/pnas.87.1.98. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Samac D. A., Hironaka C. M., Yallaly P. E., Shah D. M. Isolation and Characterization of the Genes Encoding Basic and Acidic Chitinase in Arabidopsis thaliana. Plant Physiol. 1990 Jul;93(3):907–914. doi: 10.1104/pp.93.3.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shinshi H., Neuhas J. M., Ryals J., Meins F., Jr 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. 1990 Mar;14(3):357–368. doi: 10.1007/BF00028772. [DOI] [PubMed] [Google Scholar]
- 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-beta-1,3-glucanase. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5541–5545. doi: 10.1073/pnas.85.15.5541. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Swegle M., Kramer K. J., Muthukrishnan S. Properties of Barley Seed Chitinases and Release of Embryo-Associated Isoforms during Early Stages of Imbibition. Plant Physiol. 1992 Jul;99(3):1009–1014. doi: 10.1104/pp.99.3.1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ward E. R., Uknes S. J., Williams S. C., Dincher S. S., Wiederhold D. L., Alexander D. C., Ahl-Goy P., Metraux J. P., Ryals J. A. Coordinate Gene Activity in Response to Agents That Induce Systemic Acquired Resistance. Plant Cell. 1991 Oct;3(10):1085–1094. doi: 10.1105/tpc.3.10.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wong Y. S., Maclachlan G. A. 1,3-beta-d-Glucanases from Pisum sativum Seedlings: III. DEVELOPMENT AND DISTRIBUTION OF ENDOGENOUS SUBSTRATES. Plant Physiol. 1980 Feb;65(2):222–228. doi: 10.1104/pp.65.2.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Worrall D., Hird D. L., Hodge R., Paul W., Draper J., Scott R. Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco. Plant Cell. 1992 Jul;4(7):759–771. doi: 10.1105/tpc.4.7.759. [DOI] [PMC free article] [PubMed] [Google Scholar]