Abstract
Osmotin, a pathogenesis-related antifungal protein, is relevant in induced plant immunity and belongs to the thaumatin-like group of proteins (TLPs). This article describes comparative structural and functional analysis of the two osmotin isoforms cloned from Phytophthora-resistant wild Piper colubrinum. The two isoforms differ mainly by an internal deletion of 50 amino acid residues which separates them into two size categories (16.4 kDa—PcOSM1 and 21.5 kDa—PcOSM2) with pI values 5.6 and 8.3, respectively. Recombinant proteins were expressed in E. coli and antifungal activity assays of the purified proteins demonstrated significant inhibitory activity of the larger osmotin isoform (PcOSM2) on Phytophthora capsici and Fusarium oxysporum, and a markedly reduced antifungal potential of the smaller isoform (PcOSM1). Homology modelling of the proteins indicated structural alterations in their three-dimensional architecture. Tertiary structure of PcOSM2 conformed to the known structure of osmotin, with domain I comprising of 12 β-sheets, an α-helical domain II and a domain III composed of 2 β-sheets. PcOSM1 (smaller isoform) exhibited a distorted, indistinguishable domain III and loss of 4 β-sheets in domain I. Interestingly, an interdomain acidic cleft between domains I and II, containing an optimally placed endoglucanase catalytic pair composed of Glu–Asp residues, which is characteristic of antifungal PR5 proteins, was present in both isoforms. It is well accepted that the presence of an acidic cleft correlates with antifungal activity due to the presence of endoglucanase catalytic property, and hence the present observation of significantly reduced antifungal capacity of PcOSM1 despite the presence of a strong acidic cleft, is suggestive of the possible roles played by other structural features like domain I or/and III, in deciding the antifungal potential of osmotin.
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References
Agrios, G. N. (2005). Plant pathology. Burlington: Elsevier Academic press.
Van Loon, L. C., Pierpoint, W. S., Boller, T., & Conejero, V. (1994). Recommendations for naming plant pathogenesis-related proteins. Plant Molecular Biology Reporter, 12, 245–264.
Velazhahan, R., Datta, S. K., & Muthukrishnan, S. (1999). The PR-5 family: Thaumatin-like proteins in plants. In S. K. Datta & S. Muthukrishnan (Eds.), Pathogenesis-related proteins in plants (pp. 107–129). Boca Raton: CRC press.
Roberts, W. K., & Selitrennikoff, C. P. (1990). Zeamatin, an antifungal protein from maize with membrane permeabilizing activity. Journal of General Microbiology, 136, 1771–1778.
Woloshuk, C. P., Meulenhoff, J. S., Sela-Buurlage, M., van den Elzen, P. J. M., & Cornelissen, B. J. C. (1991). Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans. Plant Cell, 3, 619–628.
Abad, L. R., D’Urzo, M. P., Liu, D., Narasimhan, M. L., Reuveni, M., Zhu, J. K., et al. (1996). Antifungal activity of tobacco osmotin has specificity and involves plasma membrane permeabilisation. Plant Science, 118, 11–23.
Liu, D., Rhodes, D., D’Urzo, M. P., Xu, Y., Narasimhan, M. L., Hasegawa, P. M., et al. (1996). In vivo and in vitro activity of truncated osmotin that is secreted into the extracellular matrix. Plant Science, 121, 123–131.
Jami, S. K., Anuradha, T. S., Guruprasad, L., & Kirti, P. B. (2007). Molecular, biochemical and structural characterization of osmotin-like protein from black nightshade (Solanum nigrum). Journal of Plant Physiology, 164, 238–252.
Liu, D., Raghothama, K. G., Hasegawa, P. M., & Bressan, R. A. (1994). Osmotin over expression in potato delays development of disease symptoms. Proceedings of the National Academy of Science of the United States of America, 91, 1888–1892.
Datta, K., Velazhahan, R., Oliva, N., Ona, I., Mew, T., & Khush, G. S. (1999). Over-expression of the cloned rice thaumatinlike protein (PR-5) gene in transgenic rice plants enhances environmently friendly resistance to Rhizoctonia solani causing sheath blight disease. Theoretical and Applied Genetics, 98, 1138–1145.
Yun, D. J., Zhao, Y., Pardo, J. M., Narasimhan, M. L., Damsz, B., & Lee, H. (1997). Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein. Proceedings of the National Academy of Science of the United States of America, 94, 7082–7087.
Anžlovar, S., Dalla Serra, M., Dermastia, M., & Menestrina, G. (1998). Membrane permeabilizing activity of pathogenesis-related protein linusitin from flax seed. Molecular Plant-Microbe Interactions, 7, 610–617.
Anžlovar, S., & Dermastia, M. (2003). The comparative analysis of osmotins and osmotin-like PR-5 proteins. Plant Biology, 5, 116–124.
Ibeas, J. I., Lee, H., Damsz, B., Prasad, D. T., Pardo, J. M., Hasegawa, P. M., et al. (2000). Fungal cell wall phosphomannans facilitate the toxic activity of a plant PR-5 protein. Plant Journal, 23, 375–383.
Narasimhan, M. L., Lee, H., Damsz, B., Singh, N. K., Ibeas, J. L., Mat-sumoto, T. K., et al. (2003). Overexpression of a cell wall glycoprotein in Fusarium oxysporum increases virulence and resistance to a plant PR-5 protein. Plant Journal, 36, 390–400.
Kadowaki, T., & Yamauchi, T. (2005). Adiponectin and adiponectin receptors. Endocrine Reviews, 26, 439–451.
Narasimhan, M. L., Coca, M. A., Jin, J., Yamauchi, T., Ito, Y., Kadowaki, T., et al. (2005). Osmotin is a homolog of mammalian adiponectin and controls apoptosis in yeast through a homolog of mammalian adiponectin receptor. Molecular Cell, 17, 171–180.
Batalia, M. A., Monzingo, A. F., Ernst, S., & Robertus, J. D. (1996). The crystal structure of the antifungal protein zeamatin, a member of the thaumatin-like, PR-5 protein family. Nature Structural Biology, 3, 19–23.
Koiwa, H., Kato, H., Nakatsu, T., Oda, J., Yamada, Y., & Sato, F. (1999). Crystal structure of tobacco PR-5d protein at 1.8 Å resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins. Journal of Molecular Biology, 286, 1137–1145.
Min, K., Ha, S. C., Hasegawa, P. M., Bressan, R. A., Yun, D., & Kim, K. K. (2004). Crystal structure of osmotin, a plant antifungal protein. Proteins: Structure, Function, and Bioinformatics, 54, 170–173.
Leone, P., Menu-Bouaouiche, L., Peumans, W. J., Payan, F., Barre, A., Roussel, A., et al. (2006). Resolution of the structure of the allergenic and antifungal banana fruit thaumatin like protein at 1.7-A°. Biochimie, 88, 45–52.
Ghosh, R., & Chakrabarti, C. (2008). Crystal structure analysis of NP24-I: A thaumatin-like protein. Planta, 228, 883–890.
Trudel, J., Grenier, J., Potvin, C., & Asselin, A. (1998). Several thaumatin-like proteins bind to β-1, 3-glucans. Plant Physiology, 118, 1431–1438.
Grenier, J., Potvin, C., Trudel, J., & Asselin, A. (1999). Some thaumatin-like proteins hydrolyse polymeric β-1, 3-glucans. Plant Journal, 19, 473–480.
Osmond, R. I., Hrmova, M., Fontaine, F., Imberty, A., & Fincher, G. B. (2001). Binding interactions between barley thaumatin-like proteins and (1, 3)-β-D-glucans. Kinetics, specificity, structural analysis and biological implications. European Journal of Biochemistry, 268, 4190–4199.
Menu-Bouaouiche, L., Vriet, C., Peumans, W. J., Barre, A., Van Damme, E. J., & Rougé, P. (2003). A molecular basis for the endo-beta 1, 3-glucanase activity of the thaumatin-like proteins from edible fruits. Biochimie, 85, 123–131.
Mani, T., & Manjula, S. (2010). Cloning and characterization of two osmotin isoforms from Piper colubrinum. Biologia Plantarum, 54, 377–380.
Vanaja, T., Neema, V. P., Mammootty, K. P., & Rajeshkumar, R. (2008). Development of a promising interspecific hybrid in black pepper (Piper nigrum L.) for Phytophthora foot rot resistance. Euphytica, 161, 437–445.
Hu, X., & Reddy, A. S. N. (1997). Cloning and expression of a PR5-like protein from Arabidopsis: Inhibition of fungal growth by bacterially expressed protein. Plant Molecular Biology, 349, 49–59.
Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry, 723, 41–74.
Broekaert, W. F., Terras, F. R. G., Cammue, B. P. A., & Vanderleyden, J. (1990). An automated quantitative assay for fungal growth inhibition. FEMS Microbiology Letters, 69, 55–60.
Salzman, R. A., Koiwa, H., Ibeas, J. I., Pardo, J. M., Hasegawa, P. M., & Bressan, R. A. (2004). Inorganic cations mediate plant PR5 protein antifungal activity through fungal Mnn1-and Mnn4-regulated cell surface glycans. Molecular Plant-Microbe Interactions, 17, 780–788.
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., et al. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acid Research, 25, 3389–33402.
Sali, A., & Blundell, T. L. (1993). Comparative protein modelling by satisfaction of spatial restraints. Journal of Molecular Biology, 234, 779–815.
Kelley, L. A., Gardner, S. P., & Sutcliffe, M. J. (1996). An automated approach for clustering an ensemble of NMR-derived protein structures into conformationally related subfamilies. Protein Engineering, 9, 1063–1065.
Sippl, M. J. (1993). Recognition of errors in three-dimensional structures of proteins. Proteins, 17, 355–362.
Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew, R. K., et al. (1998). Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function. Journal of Computational Chemistry, 19, 1639–1662.
Kouwizjer, M. L. C. E., & Grootenhuis, P. D. J. (1995). Parametrization and application of CHEAT95, an extended atom force field for hydrated oligosaccharides. Journal of Physical Chemistry, 99, 13426–13436.
Aswati Nair, R., Kiran, A. G., Sivakumar, K. C., & Thomas, G. (2010). Molecular characterization of an oomycete-responsive PR-5 protein gene from Zingiber zerumbet. Plant Molecular Biology Reporter, 28, 128–135.
Chan, Y. W., Tung, W. L., Griffith, M., & Chow, K. C. (1999). Cloning of a cDNA encoding the thaumatin-like protein of winter rye (Secale cereale L. Musketeer) and its functional characterisation. Journal of Experimental Botany, 50, 627–1628.
Thevissen, K., Osborn, R. W., Acland, D. P., & Broekaert, W. F. (1997). Specific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes. Journal of Biological Chemistry, 272, 32176–32181.
Thevissen, K., Osborn, R. W., Acland, D. P., & Broekaert, W. F. (2000). Specific binding sites for an antifungal plant defensin from Dahlia (Dahlia merckii) on fungal cells are required for antifungal activity. Molecular Plant-Microbe Interactions, 13, 54–61.
Veronese, P., Ruiz, M. T., Coca, M. A., Hernandez-Lopez, A., Lee, H., Ibeas, J. I., et al. (2003). In defense against pathogens both plant sentinels and foot soldiers need to know the enemy. Plant Physiology, 131, 1580–1590.
Hutchins, K., & Bussey, H. (1983). Cell wall receptor for yeast killer toxin: Involvement of (1–6)-β-d-glucan. Journal of Bacteriology, 1549, 161–169.
Bussey, H. (1991). K1 killer toxin, a pore-forming protein from yeast. Molecular Microbiology, 5, 2339–23431.
Acknowledgments
Authors would like to thank Dr. N. Anith, Kerala Agricultural University, Vellayani, Trivandrum for the fungal strains. T.M. would like to acknowledge Council for Scientific and Industrial Research, New Delhi, Government of India, for CSIR-Junior Research Fellowship, and MS gratefully acknowledges the Department of Biotechnology, Government of India, for financial support in the form of research grant.
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Mani, T., Sivakumar, K.C. & Manjula, S. Expression and Functional Analysis of Two Osmotin (PR5) Isoforms with Differential Antifungal Activity from Piper colubrinum: Prediction of Structure–Function Relationship by Bioinformatics Approach. Mol Biotechnol 52, 251–261 (2012). https://doi.org/10.1007/s12033-011-9489-0
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DOI: https://doi.org/10.1007/s12033-011-9489-0