Skip to main content
SpringerLink
Log in

Overexpression of Camellia sinensis Thaumatin-Like Protein, CsTLP in Potato Confers Enhanced Resistance to Macrophomina phaseolina and Phytophthora infestans Infection

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Thaumatin-like proteins (TLPs), a class of pathogenesis related proteins are induced in response to pathogens and exhibit antifungal property when overexpressed in transgenic plants. In the present study, we have raised transgenic potato plants overexpressing TLP gene of Camellia sinensis (CsTLP). Fungal resistance assays of transgenic potato elucidated the potential role of CsTLP in imparting tolerance to fungal pathogens, Macrophomina phaseolina (necrotrophic) and Phytophthora infestans (hemi-biotrophic). Transgenic tubers with higher resistance to M. phaseolina, showed a concomitant and significant increase in transcripts of StPAL, StLOX, and StTLP genes involved in phenylpropanoid, lipoxygenase, and general defense response pathway, respectively after infection. Importantly, leaves of CsTLP transgenic lines inoculated with P. infestans spores under in vitro conditions also showed a resistant phenotype. The resistant phenotype recorded for the two important fungal pathogens by CsTLP transgenic potato plants is remarkable, since no effective control methods and no resistant cv. against M. phaseolina has been identified so far in potato.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

PR:

Pathogenesis related

TLP:

Thaumatin-like protein

PAL:

Phenyl-alanine ammonia lyase

LOX:

Lipoxygenase

CsTLP:

Camellia sinensis thaumatin-like protein

References

  1. Fry, W. E., Goodwin, E., Dyer, A. T., Matuszak, J. M., Drenth, A., Tooley, P. W., et al. (1993). Historical and recent migrations of Phytophthora infestans: Chronology pathways and implications. Plant Disease, 77, 653–661.

    Article  Google Scholar 

  2. Suzuki, N., Rizhsky, L., Linag, H., Shuman, J., Shulaev, V., & Mittler, R. (2005). Enhanced tolerance to environmental stress in transgenic plants expressing the transcriptional coactivator multiprotein bridging factor 1c. Plant Physiology, 139, 1313–1322.

    Article  CAS  Google Scholar 

  3. Smith, C. J. (1996). Accumulation of phytoalexins: Defense mechanism and stimulus response system. New Phytologist, 132, 1–45.

    Article  CAS  Google Scholar 

  4. Dixon, R. A. (2005). Engineering of plant natural product pathways. Current Opinion in Plant Biology, 8, 329–336.

    Article  CAS  Google Scholar 

  5. Dixon, R. A. (2011). Chris Lamb: A visionary leader in plant science. Annual Review of Phytopathology, 49, 31–45.

    Article  CAS  Google Scholar 

  6. Blée, E. (1998). Phytoxylipins and plant defense reactions. Progress in Lipid Research, 37, 33–72.

    Article  Google Scholar 

  7. Feussner, I., & Wasternack, C. (2002). The lipoxygenase pathway. Annual Review of Plant Physiology and Plant Molecular Biology, 53, 275–297.

    Article  CAS  Google Scholar 

  8. van Loon, L. C., Rep, M., & Pieterse, C. M. J. (2006). Significance of inducible defense-related proteins in infected plants. Annual Review of Phytopathology, 44, 135–162.

    Article  Google Scholar 

  9. Lawton, M. A., & Lamb, C. J. (1987). Transcriptional activation of plant defense genes by fungal elicitor, wounding and infection. Molecular and Cellular Biology, 7, 335–341.

    CAS  Google Scholar 

  10. Dixon, R. A. (2001). Natural products and disease resistance. Nature, 411, 843–847.

    Article  CAS  Google Scholar 

  11. Saunders, J., & O’neill, N. (2004). The characterization of defense responses to fungal infection in alfalfa. Biocontrol, 49, 715–728.

    Article  CAS  Google Scholar 

  12. Menè-Saffrané, L., Esquerré-Tugayé, M., & Fournier, J. (2003). Constitutive expression of an inducible lipoxygenase in transgenic tobacco decreases susceptibility to Phytophthora parasitica var. nicotianae. Molecular Breeding, 12, 271–282.

    Article  Google Scholar 

  13. Zhao, J., Davis, L. C., & Verporte, R. (2005). Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances, 23, 283–333.

    Article  CAS  Google Scholar 

  14. Gardner, H. W. (1991). Recent investigations into the lipoxygenase pathway in plants. Biochimica et Biophysica Acta, 1084, 221–239.

    Article  CAS  Google Scholar 

  15. Bate, N. J., Orr, N., Ni, W., Meromi, A., Nadler-Hassar, T., Doerner, P. W., et al. (1994). Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis. Proceedings of the National Academy of Sciences of United States of America, 91, 7608–7612.

    Article  CAS  Google Scholar 

  16. Howles, P. A., Sewalt, V. J. H., Paiva, N. L., Elkind, Y., Bate, N. J., Lamb, C., et al. (1996). Overexpression of l-phenylalanine ammonia-lyase in transgenic tobacco plants reveals control points for flux into phenylpropanoid biosynthesis. Plant Physiology, 112, 1617–1624.

    CAS  Google Scholar 

  17. Hain, R., Reif, H. J., Krause, E., Langebartels, R., & Kindl, H. (1993). Disease resistance results from foreign phytoalexin expression in a novel plant. Nature, 361, 153–156.

    Article  CAS  Google Scholar 

  18. Shadle, G. L., Wesley, S. V., Korth, K. K., Chen, F., Lamb, C., & Dixon, R. A. (2003). Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of l-phenylalanine ammonia-lyase. Phytochemistry, 64, 153–161.

    Article  CAS  Google Scholar 

  19. Göbel, C., Feussner, I., Schimidt, A., Scheel, D., Sanchez-Serrano, J., Hamberg, M., et al. (2001). Oxylipin profiling reveals the preferential stimulation of the 9-lipoxygenase pathway in elicitor-treated potato cells. The Journal of Biological Chemistry, 276, 6273–6627.

    Article  Google Scholar 

  20. Monteiro, S., Barakat, M., Piçarra-Pereira, M. A., Teixeira, A. R., & Ferreira, R. B. (2003). Osmotin and thaumatin from grape: A putative general defense mechanism against pathogenic fungi. Phytopathology, 93, 1505–1512.

    Article  CAS  Google Scholar 

  21. Aguilar, I., Poza-Carrin, C., Gui, A., & Rodrguez-Palenzuela, P. (2002). Erwinia chrysanthemi genes specifically induced during infection in chicory leaves. Molecular Plant Pathology, 3, 271–275.

    Article  CAS  Google Scholar 

  22. de León, I. P., Oliver, J. P., Castro, A., Gaggero, C., Bentancor, M., & Vidal, S. (2007). Erwinia carotovora elicitors and Botrytis cinerea activate defense responses in Physcomitrella patens. BMC Plant Biology, 7, 52–63.

    Article  Google Scholar 

  23. Fidantsef, A. L., & Bostock, R. M. (1998). Characterization of potato tuber lipoxygenase cDNAs and lipoxygenase expression in potato tubers and leaves. Physiologia Plantarum, 102, 257–271.

    Article  CAS  Google Scholar 

  24. Jayaraj, J., Muthukrishnan, S., Liang, G. H., & Velazhahan, R. (2004). Jasmonic acid and salicylic acid induce accumulation of β-1,3-glucanase and thaumatin-like proteins in wheat and enhance resistance against Stagonospora nodorum. Biologia Plantarum, 48, 425–430.

    Article  CAS  Google Scholar 

  25. Kariola, T., Palomäki, T. A., Brader, G., & Palva, E. T. (2003). Erwinia carotovora subsp. carotovora and Erwinia-derived elicitors HrpN and PehA trigger distinct but interacting defense responses and cell death in Arabidopsis. Molecular Plant Microbe Interaction, 16, 179–187.

    Article  CAS  Google Scholar 

  26. Ramamoorthy, V., Raguchander, T., & Samiyappan, R. (2002). Induction of defense-related proteins in tomato roots treated with Pseudomonas fluorescens Pf1 and Fusarium oxysporum f. sp. lycopersici. Plant and Soil, 239, 55–68.

    Article  CAS  Google Scholar 

  27. Cornelissen, B. J. C., & Melchers, L. S. (1993). Strategies for control of fungal diseases with transgenic plants. Plant Physiology, 101, 709–712.

    CAS  Google Scholar 

  28. Punja, Z. K. (2001). Genetic engineering of plants to enhance resistance to fungal pathogens—A review of progress and future prospects. Canadian Journal of Plant Pathology, 23, 216–235.

    Article  CAS  Google Scholar 

  29. Rao, G. U., Kaur, M., Verma, A., Sihachakr, D., & Rajam, M. V. (1999). Genetic engineering of crop plants for resistance to fungal pathogens. Journal of Plant Biology, 26, 31–42.

    Google Scholar 

  30. Cornelissen, B. J. C., Hooft Van Huijsduijnen, R. A., & Bol, J. F. (1986). A tobacco mosaic virus-induced tobacco protein is homologous to the sweet tasting protein, thaumatin. Nature, 231, 531–532.

    Article  Google Scholar 

  31. Chu, K. T., & Ng, T. B. (2003). Isolation of a large thaumatin-like antifungal protein from seeds of the Kweilin chestnut Castanopsis chinensis. Biochemical and Biophysical Research Communications, 301, 364–370.

    Article  CAS  Google Scholar 

  32. 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.

    Article  CAS  Google Scholar 

  33. Wang, Q., Li, F., Zhang, X., Zhang, Y., Hou, Y., Zhang, S., et al. (2011). Purification and characterization of a CkTLP protein from Cynanchum komarovii seeds that confers antifungal activity. PLoS ONE, 6, e16930.

    Article  CAS  Google Scholar 

  34. Liu, D., Raghothama, K. G., Hasegawa, P. M., & Bressan, R. A. (1994). Osmotin overexpression in potato delays development of disease symptoms. Proceedings of the National Academy of Sciences of United States of America, 91, 1888–1892.

    Article  CAS  Google Scholar 

  35. Zhu, B., Chen, T. H. H., & Li, P. H. (1996). Analysis of late-blight disease resistance and freezing tolerance in transgenic potato plants expressing sense and antisense genes for an osmotin-like protein. Planta, 198, 70–77.

    Article  CAS  Google Scholar 

  36. Velazhahan, R., & Muthukrishnan, S. K. (2003). Transgenic tobacco plants constitutively overexpressing a rice thaumatin-like protein (PR-5) show enhanced resistance to Alternaria alternata. Biologia Plantarum, 47, 347–354.

    Article  CAS  Google Scholar 

  37. Datta, K., Velazhahan, R., Oliva, N., Mew, T., Khush, G. S., Muthukrishnan, S., et al. (1999). Over-expression of cloned rice thaumatin-like-protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease. Theoretical and Applied Genetics, 98, 1138–1145.

    Article  CAS  Google Scholar 

  38. Chen, W. P., Chen, P. D., Liu, D. J., Kjnost, R., Friebe, B., Velazhahan, R., et al. (1999). Development of wheat scab symptoms is delayed in transgenic wheat plants that constitutively express a rice thaumatin-like protein gene. Theoretical and Applied Genetics, 99, 755–760.

    Article  CAS  Google Scholar 

  39. Anand, A., Zhou, T., Trick, H. N., Gill, B. S., & Bockus, W. W. (2003). Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum. Journal of Experimental Botany, 54, 1101–1111.

    Article  CAS  Google Scholar 

  40. Thompson, C. E., Fernandes, C. L., De-Souza, O. N., Salzano, F. M., Bonatto, S. L., & Freitas, L. B. (2007). Molecular modelling of pathogenesis-related proteins of family 5. Cell Biochemistry and Biophysics, 44, 385–394.

    Article  Google Scholar 

  41. Seo, P. J., Lee, A. K., Xiang, F., & Park, C. M. (2008). Molecular and functional profiling of Arabidopsis pathogenesis-related genes: Insights into their roles in salt response of seed germination. Plant and Cell Physiology, 49, 334–344.

    Article  CAS  Google Scholar 

  42. Sakamoto, Y., Watanabe, H., Nagai, M., Nakade, K., & Takahashi, M. (2006). Lentinula edodes tlg1 encodes a thaumatin-like protein that is involved in lentinan degradation and fruiting body senescence. Plant Physiology, 141, 793–801.

    Article  CAS  Google Scholar 

  43. Ladyzhenskaia, E. P., & Korableva, N. P. (2006). The effect of thaumatin gene overexpression on the properties of H+-ATPase from the plasmalemma of potato tuber cells. Applied Biochemistry and Microbiology, 42, 409–413.

    Article  Google Scholar 

  44. Rajam, M. V., Chandola, N., Goud, P. S., Singh, D., Kashyap, V., Choudhary, M. L., et al. (2007). Thaumatin gene confers resistance to fungal pathogens as well as tolerance to abiotic stresses in transgenic tobacco plants. Biologia Plantarum, 51, 135–141.

    Article  CAS  Google Scholar 

  45. Van Haute, E., Joos, H., Maes, S., Warren, G., Van Montagu, M., & Schell, J. (1983). Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: A novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO Journal, 2, 411–418.

    Google Scholar 

  46. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473–497.

    Article  CAS  Google Scholar 

  47. Doyle, J. J., & Doyle, J. L. (1990). Isolation of plant genomic DNA from fresh tissue. Focus, 12, 13–15.

    Google Scholar 

  48. Ghawana, S., Paul, A., Kumar, H., Kumar, A., Singh, H., Bhardwaj, P. K., et al. (2011). An RNA isolation system for plant tissues rich in secondary metabolites. BMC Research Notes, 4, 85.

    Article  CAS  Google Scholar 

  49. Singh, K., Raizada, J., Bhardwaj, P., Ghawana, S., Rani, A., Singh, H., et al. (2004). 26S rRNA-based internal control gene primer pair for reverse transcription polymerase chain reaction-based quantitative expression studies in diverse plant species. Analytical Biochemistry, 335, 330–333.

    Article  CAS  Google Scholar 

  50. White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gel-fand, J. J. Shinsky, & T. J. White (Eds.), PCR protocols: A guide to methods and applications (pp. 315–322). New York: Academic Press.

    Google Scholar 

  51. 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 Acids Research, 25, 3389–3402.

    Article  CAS  Google Scholar 

  52. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G., et al. (2003). Multiple sequence alignment with the clustal series of programs. Nucleic Acids Research, 31, 3497–3500.

    Article  CAS  Google Scholar 

  53. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111–120.

    Article  CAS  Google Scholar 

  54. Rozen, S., & Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. Methods in Molecular Biology, 132, 365–386.

    CAS  Google Scholar 

  55. Ducreux, L. J. M., Morris, W. L., Hedley, P. E., Shepherd, T., Davies, H. V., Millam, S., et al. (2005). Taylor metabolic engineering of high carotenoid potato tubers containing enhanced levels of β-carotene and lutein. Journal of Experimental Botany, 56, 81–89.

    CAS  Google Scholar 

  56. Pfaffl, M. W., Horgan, G. W., & Dempfle, L. (2002). Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Research, 30, e36.

    Article  Google Scholar 

  57. El-kereamy, A., El-sharkawy, I., Ramamoorthy, R., Taheri, A., Errampalli, D., Kumar, P., et al. (2011). Prunus domestica pathogenesis-related protein-5 activates the defense response pathway and enhances the resistance to fungal infection. PLoS ONE, 6, e17973.

    Article  CAS  Google Scholar 

  58. Dhingra, O. K., & Sinclair, J. B. (1978). Biology and pathology of Macrophomina phaseolina. Vicosa: Imprensa Universitariam Universidade federal De Vicosa.

    Google Scholar 

  59. Punja, Z. K. (2005). Transgenic carrots expressing a thaumatin-like protein display enhanced resistance to several fungal pathogens. Canadian Journal of Plant Pathology, 27, 291–296.

    Article  CAS  Google Scholar 

  60. Singh, N. K., Bracker, C. A., Hasegawa, P. M., Handa, A. K., Buckel, S., Hermodson, M. A., et al. (1987). Characterization of osmotin: A thaumatin-like protein associated with osmotic adaptation in plant cells. Plant Physiology, 85, 529–536.

    Article  CAS  Google Scholar 

  61. Kumar, V., Parkhi, V., & Kenerley, C. M. (2009). Defense-related gene expression and enzyme activities inn transgenic cotton plants expressing an endochitinase gene from Trichoderma virens in response to interaction with Rhizoctonia solani. Planta, 230, 277–291.

    Article  CAS  Google Scholar 

  62. Lindqvist-Kreuze, H., Carbajulca, D., Gonzalez-Escobedo, G., Pérez, W., & Bonierbale, M. (2010). Comparison of transcript profiles in late blight-challenged Solanum cajamarquense and B3C1 potato clones. Molecular Plant Pathology, 11, 513–530.

    Article  CAS  Google Scholar 

  63. Linthorst, H. J. M., Meuwissen, R. L. J., Kauffmann, S., & Bol, J. F. (1989). Constitutive expression of pathogenesis-related proteins PR-1, GRP, and PR-S in tobacco has no effect on virus infection. Plant Cell, 1, 285–291.

    CAS  Google Scholar 

  64. Yoshioka, H., Miyabe, M., Hayakawa, Y., & Doke, N. (1996). Expression of genes for phenylalanine ammonia-lyase and 3-hydroxy-3-methylglutaryl CoA reductase in aged potato tubers infected with Phytophthora infestans. Plant and Cell Physiology, 37, 81–90.

    Article  CAS  Google Scholar 

  65. Kröner, A., Hamelin, G., Andrivon, D., & Val, F. (2011). Quantitative resistance of potato to Pectobacterium atrosepticum and Phytophthora infestans: Integrating PAMP-triggered response and pathogen growth. PLoS ONE, 6, e23331.

    Article  Google Scholar 

  66. Yang, Z., Cramer, C. L., & Lacy, G. H. (1989). System for simultaneous study of bacterial and plant genes in soft rot of potato. Molecular Plant Microbe Interactions, 2, 195–201.

    Article  Google Scholar 

  67. Bell, E., & Mullet, J. E. (1991). Lipoxygenase gene expression is modulated in plants by water deficit, wounding, and methyl jasmonate. Molecular and General Genetics, 230, 456–462.

    Article  CAS  Google Scholar 

  68. Melan, M. A., Dong, X., Endara, M. E., Davis, K. R., Ausubel, F. M., & Peterman, T. K. (1993). An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens, abscisic acid, and methyl jasmonate. Plant Physiology, 101, 441–450.

    Article  CAS  Google Scholar 

  69. Kolomietes, M. V., Chen, H., Gladon, R. J., Braun, E. J., & Hannapel, D. J. (2000). A leaf lipoxygenase of potato induced specifically by pathogen infection. Plant Physiology, 124, 1121–1130.

    Article  Google Scholar 

  70. Gao, X., Starr, J., Göbel, C., Engelberth, J., Feussner, I., Tumlinson, J., et al. (2008). Maize 9-lipoxygenase ZmLOX3 controls development, root-specific expression of defense genes, and resistance to root-knot nematodes. Molecular Plant Microbe Interaction, 21, 98–109.

    Article  CAS  Google Scholar 

  71. Véronési, C., Rickauer, M., Fournier, J., Pouénat, M. L., & Esquerré-Tugayé, M. T. (1996). Lipoxygenase gene expression in the tobacco–Phytophthora parasitica nicotianae interaction. Plant Physiology, 112, 997–1004.

    Article  Google Scholar 

  72. Gao, X., Brodhagen, M., Isakeit, T., Brown, S. H., Göbel, C., Betran, J., et al. (2009). Inactivation of the lipoxygenase ZmLOX3 increase susceptibility of maize to Aspergillus spp. Molecular Plant Microbe Interaction, 22, 222–231.

    Article  Google Scholar 

Download references

Acknowledgments

Karan Acharya gratefully acknowledges Council of Scientific and Industrial Research (CSIR), New Delhi, India for research fellowship and thanks to Dr. Tejpal Gill and Pravin Rahi for thoughtful discussions and advice. We thank A. Paul for providing the pCAMBIA-CsTLP construct. This research was financially supported by CSIR, New Delhi, India (under project NWP0020). Acknowledgement has also been given to Dr. S.K. Chakorborty, Division of crop protection, CPRI, Shimla, India for providing cultures of P. infestans. Manuscript represents IHBT communication number 3299.

Author information

Authors and Affiliations

  1. Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur, 176 061, HP, India

    Karan Acharya, Awadhesh K. Pal, Sanjay Kumar, Anil K. Singh & Paramvir S. Ahuja

  2. Hill Area Tea Science Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, 176 061, HP, India

    Arvind Gulati

Authors
  1. Karan Acharya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Awadhesh K. Pal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arvind Gulati
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sanjay Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anil K. Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Paramvir S. Ahuja
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paramvir S. Ahuja.

Electronic supplementary material

Below is the link to the electronic supplementary material.

12033_2012_9603_MOESM1_ESM.doc

Fig. S1: Alignment of amino acid sequence of Nicotiana tabacum osmotin (Nt-Osmotin, AAA34089) with C. sinensis thaumatin-like protein (CsTLP, ABE01396) using ClustalW. (DOC 308 kb). Supplementary material 1 (DOC 308 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Acharya, K., Pal, A.K., Gulati, A. et al. Overexpression of Camellia sinensis Thaumatin-Like Protein, CsTLP in Potato Confers Enhanced Resistance to Macrophomina phaseolina and Phytophthora infestans Infection. Mol Biotechnol 54, 609–622 (2013). https://doi.org/10.1007/s12033-012-9603-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-012-9603-y

Keywords

Search

Navigation