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Expression profiling of a complex thaumatin-like protein family in western white pine


The protein content in the plant apoplast is believed to change dramatically as a result of host defense response upon infection with various pathogens. In this study, six novel thaumatin-like proteins (TLPs) were identified in western white pine (Pinus monticola) needle apoplast by a proteomic strategy using two-dimensional protein electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sequent cDNA cloning found that ten P. monticola TLP genes (PmTLP-L1 to -L6 and -S1 to -S4) were expressed in various tissues. Phylogenetic analysis demonstrated that these PmTLP genes belong to a large, complex, and highly diverse plant TLP family. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) using gene-specific primer pairs showed that each PmTLP gene exhibited a characteristic pattern of mRNA expression based on their unique organ distribution, seasonal regulation, and response to abiotic and biotic stresses. A time-course analysis at the early stages of infection by white pine blister rust pathogen Cronartium ribicola revealed that a coordinated upregulation of multiple PmTLP genes was involved in P. monticola major gene (Cr2) resistance. The structural and expressional differentiations suggest that the PmTLP family may contribute to host defense as well as other mechanism.

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Hypersensitive response


Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry


Pinus monticola thaumatin-like protein




Rapid amplification of cDNA ends


Quantitative reverse transcription-polymerase chain reaction


White pine blister rust


  1. Abad LR, D’Urzo MP, Liu D, Narasimhan ML, Reuveni M, Zhu JK, Niu X, Singh NK, Hasegawa PM, Bressen RA (1996) Antifungal activity of tobacco osmotin has specificity and involves plasma membrane permeabilization. Plant Sci 118:11–23

  2. Alonso P, Cortizo M, Cantón FR, Fernández B, Rodríguez A, Centeno ML, Cánovas FM, Ordás RJ (2007) Identification of genes differentially expressed during adventitious shoot induction in Pinus pinea cotyledons by subtractive hybridization and quantitative PCR. Tree Physiol 27:1721–1730

  3. Antikainen M, Griffith M (1997) Antifreeze protein accumulation in freezing-tolerant cereals. Physiol Plant 99:423–432

  4. Batalia MA, Monzingo AF, Ernst S, Roberts W, Robertus JD (1996) The crystal structure of the antifungal protein zeamatin, a member of the thaumatin-like, PR-5 protein family. Nat Struct Biol 3:19–23

  5. Brandazza A, Angeli S, Tegoni M, Cambillau C, Pelosi P (2004) Plant stress proteins of the thaumatin-like family discovered in animals. FEBS Lett 572:3–7

  6. Brito N, Espino JJ, Gonzalez C (2006) The endo-β-1, 4-xylanase Xyn11A is required for virulence in Botrytis cinerea. Mol Plant Microbe Interact 19:25–32

  7. Christensen AB, Cho BH, Naesby M, Gregersen PL, Brandt J, Madri-Ordenãna K, Collinge DB, Thordal-Christensen H (2002) The molecular characterization of two barley proteins establishes the novel PR-17 family of pathogenesis-related proteins. Mol Plant Pathol 3:135–144

  8. Colditz F, Niehaus K, Krajinski F (2007) Silencing of PR-10-like proteins in Medicago truncatula results in an antagonistic induction of other PR proteins and in an increased tolerance upon infection with the oomycete Aphanomyces euteiches. Planta 226:57–71

  9. D’Angeli S, Altamura MM (2007) Osmotin induces cold protection in olive trees by affecting programmed cell death and cytoskeleton organization. Planta 225:1147–1163

  10. Davidson J, Ekramoddoullah AKM (1997) Analysis of bark proteins in blister rust-resistant and susceptible western white pine (Pinus monticola). Tree Physiol 17:663–669

  11. Ekramoddoullah AKM, Tan Y (1998) A modification for the improved analysis of differential protein accumulation by two-dimensional polyacrylamide gel electrophoresis. Phytochem Anal 9:159–161

  12. Ekramoddoullah AKM, Liu JJ, Zamani A (2006) Cloning and characterization of an anti-fungal peptide gene (Pm-AMP1) in Pinus monticola (Douglas Ex D. Don). Phytopathology 96:164–170

  13. Elvira MI, Galdeano MM, Gilardi P, García-Luque I, Serra MT (2008) Proteomic analysis of pathogenesis-related proteins (PRs) induced by compatible and incompatible interactions of pepper mild mottle virus (PMMoV) in Capsicum chinense L3 plants. J Exp Bot 59:1253–1265

  14. Fierens E, Rombouts S, Gebruers K, Goesaert H, Brijs K, Beaugrand J, Volckaert G, Van Campenhout S, Proost P, Courtin CM, Delcour JA (2007) TLXI, a novel type of xylanase inhibitor from wheat (Triticum aestivum) belonging to the thaumatin family. Biochem J 403(Pt 3):583–591

  15. Futamura N, Tani N, Tsumura Y, Nakajima N, Sakaguchi M, Shinohara K (2005) Characterization of genes for novel thaumatin-like proteins in Cryptomeria japonica. Tree Physiol 26:51–62

  16. Ghosh R, Chakrabarti C (2008) Crystal structure analysis of NP24-I: a thaumatin-like protein. Planta 228:883–890

  17. Grenier J, Potvin C, Asselin A (2000) Some fungi express β-1, 3-glucanases similar to thaumatin-like proteins. Mycologia 92:841–848

  18. Hoffmann-Sommergruber K (2002) Pathogenesis-related (PR)-proteins identified as allergens. Biochem Soc Trans 30:930–935

  19. Jones JDG, Dangl JL (2006) The plant immune system. Nature 444:323–329

  20. Kaneko R, Kitabatake N (2001) Structure-sweetness relationship in thaumatin: importance of lysine residues. Chem Senses 26:167–177

  21. Kinloch BB Jr (2003) White pine blister rust in North America: past and prognosis. Phytopathology 93:1044–1047

  22. Kinloch BB Jr, Sniezko RA, Barnes GD, Greathouse TE (1999) A major gene for resistance to white pine blister rust in western white pine from the Western Cascade Range. Phyotpathol 89:861–867

  23. Kinloch BB Jr, Sniezko RA, Dupper GE (2003) Origin and distribution of Cr2, a gene for resistance to white pine blister rust in natural populations of western white pine. Phytopathology 93:691–694

  24. Kitajima S, Sato F (1999) Plant pathogenesis-related proteins: molecular mechanisms of gene expression and protein function. J Biochem (Tokyo) 125:1–8

  25. 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. J Mol Biol 286:1137–1145

  26. Krebitz M, Wagner B, Ferreira F, Peterbauer C, Campillo N, Witty M, Kolarich D, Steinkellner H, Scheiner O, Breiteneder H (2003) Plant-based heterologous expression of Mal D 2, a thaumatin-like protein and allergen of apple (Malus domestica), and its characterization as an antifungal protein. J Mol Biol 329:721–730

  27. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163

  28. Kuwabara C, Takezawa D, Shimada T, Hamada T, Fujikawa S, Arakawa K (2002) Abscisic acid- and cold-induced thaumatin-like protein in winter wheat has antifungal activity against snow mould, Microdochium nivale. Physiol Plant 115:101–110

  29. Léon-Kloosterziel KM, Verhagen BWM, Keurentjes JJB, Van Pelt JA, Rep M, Van Loon LC, Pieterse CMJ (2005) Colonization of the Arabidopsis rhizosphere by fluorescent Pseudomonas spp. activates a root-specific, ethylene-responsive PR-5 gene in the vascular bundle. Plant Mol Biol 57:731–748

  30. Liu J-J, Ekramoddoullah AKM (2003) Root-specific expression of a western white pine PR10 gene is mediated by different promoter regions in transgenic tobacco. Plant Mol Biol 52:103–120

  31. Liu J-J, Ekramoddoullah AKM (2004) Characterization, expression and evolution of two novel subfamilies of Pinus monticola (Dougl. Ex D. Don) cDNAs encoding pathogenesis-related (PR)-10 proteins. Tree Physiol 24:1377–1385

  32. Liu J-J, Ekramoddoullah AKM (2007) The CC-NBS-LRR subfamily in western white pine (Pinus monticola D. Don.): targeted identification, gene expression and genetic linkage with disease resistance against Cronartium ribicola. Phytopathology 97:728–736

  33. Liu J-J, Ekramoddoullah AKM (2008) Development of LRR polymorphism, AFLP, and SCAR markers to the Cronartium ribicola resistance gene Cr2 in western white pine (Pinus monticola). Tree Genet Genomes 4:601–610

  34. Liu J-J, Ekramoddoullah AKM, Yu X (2003) Differential expression of multiple PR10 proteins in western white pine following wounding, fungal infection and cold-hardening. Physiol Plant 119:544–553

  35. Liu J-J, Hunt RS, Ekramoddoullah AKM (2004) Recent insights into western white pine genetic resistance to white blister rust. Recent Res Dev Biotech Bioeng 6:65–76

  36. Liu J-J, Ekramoddoullah AKM, Zamani A (2005) A class IV chitinase is up-regulated upon fungal infection and abiotic stresses and associated with slow-canker-growth resistance to Cronartium ribicola in western white pine (Pinus monticola, Dougl. Ex D. Don). Phytopathology 95:284–291

  37. Maloy OC (1997) White pine blister rust control in North America: a case history. Annu Rev Phytopathol 35:87–109

  38. Min K, Ha SC, Hasegawa PM, Bressan RA, Yun D-J, Kim KK (2004) Crystal structure of osmotin, a plant antifungal protein. Proteins 54:170–173

  39. Mylona P, Moerman M, Yang W-C, Gloudemans T, Van De Kerckhove J, van Kammen A, Bisseling T, Franssen HJ (1994) The root epidermis-specific pea gene RH2 is homologous to a pathogenesis-related gene. Plant Mol Biol 26:39–50

  40. Nogueira FTS, De Rosa VE Jr, Menossi M, Ulian EC, Arruda P (2003) RNA expression profiles and data mining of sugarcane response to low temperature. Plant Physiol 132:1811–1824

  41. O’Leary SJ, Poulis BA, von Aderkas P (2007) Identification of two thaumatin-like proteins (TLPs) in the pollination drop of hybrid yew that may play a role in pathogen defence during pollen collection. Tree Physiol 27:1649–1659

  42. Osmond RIW, Hrmova M, Fontaine F, Imberty A, Fincher GB (2001) Binding interactions between barley thaumatin-like proteins an (1, 3)-β-d-glucans. Eur J Biochem 15:4190–4199

  43. Pfaffl MW, Horgan GH, Dempfle L (2002) Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucl Acids Res 30:e36

  44. Piggott N, Ekramoddoullah AKM, Liu J-J, Yu X (2004) Gene cloning and expression of a thaumatin-like protein of western white pine (Pinus monticola D.Don). Physiol Mol Plant Pathol 64:1–8

  45. Ralph SG, Yueh H, Friedmann M, Aeschliman D, Zeznik JA, Nelson CC, Butterfield YS, Kirkpatrick R, Liu J, Jones SJ, Marra MA, Douglas CJ, Ritland K, Bohlmann J (2006) Conifer defense against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large scale changes of the host transcriptome. Plant Cell Environ 29:1545–1570

  46. Regalado AP, Ricardo CPP (1996) Study of intercellular fluid in healthy Lupinus albus organs. Plant Physiol 110:227–232

  47. Rep M, Dekker HL, Vossen JH, de Boer AD, Houterman PM, Speijer D, Back JW, de Koster CG, Cornelissen BJC (2002) Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato. Plant Physiol 130:904–917

  48. Roberts WK, Selitrennikoff CP (1990) Zeamatin, an antifungal protein from maize with membrane-permeabilizing activity. J Gen Microbiol 136:1771–1778

  49. Sakamoto Y, Watanabe H, Nagai M, Nakade K, Takahashi M, Sato T (2006) Lentinula edodes tlg1 encodes a thaumatin-like protein that is involved in Lentinan degradation and fruiting body senescence. Plant Physiol 141:793–801

  50. Selitrennikoff CP (2001) Antifungal proteins. Appl Environ Microbiol 67:2883–2894

  51. Shatters RG Jr, Boykin LM, Lapointe SL, Hunter WB, Weathersbee AA 3rd (2006) Phylogenetic and structural relationships of the PR5 gene family reveal an ancient multigene family conserved in plants and select animal taxa. J Mol Evol 63:12–29

  52. Shevchenko A, Sunyaev S, Loboda A, Shevchenko A, Bork P, Ens W, Standing K (2001) Charting the proteomes of organisms with unsequenced genomes by MALDI-Quadrupole Time-of-Flight mass spectrometry and BLAST homology searching. Anal Chem 73:1917–1926

  53. Shiu S-H, Bleecker AB (2001) Plant receptor-like kinase gene family: diversity, function, and signaling. Sci STKE 113:re22

  54. Singh NK, Nelson DE, Kuhn D, Hasegawa PM, Bressan PA (1989) Molecular cloning of osmotin and regulation of its expression by ABA and adaptation to low water potential. Plant Physiol 90:1096–1101

  55. Sniezko RA (2006) Resistance breeding against nonnative pathogens in forest trees—current successes in North America. Can J Pathol 28:S270–S279

  56. van der Wel H, Loewe K (1972) Isolation and characterization of thaumatin I and II, the sweet-tasting proteins from Thaumatococcus daniellii Benth. Eur J Biochem 31:221–225

  57. van Loon LC, Rep M, Pieterse CMJ (2006) Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 44:135–162

  58. Velazhahan R, Datta SK, Muthukrishnan S (1999) The PR-5 family: thaumatin-like proteins in plants. In: Datta SK, Muthukrishnan S (eds) Pathogenesis related proteins in plants. CRC Press, Boca Raton, pp 107–129

  59. Wang L, Duman JG (2006) A thaumatin-like protein from larvae of the beetle Dendroides canadensis enhances the activity of antifreeze proteins. Biochemistry 45:1278–1284

  60. Wang X, Zafian P, Choudhary M, Lawton M (1996) The PR5 K receptor protein kinase from Arabidopsis thaliana is structurally related to a family of plant defense proteins. Proc Natl Acad Sci USA 93:2598–2602

  61. Yu XM, Griffith M (1999) Antifreeze proteins in winter rye leaves form oligomeric complexes. Plant Physiol 119:1361–1369

  62. Zamani A, Sturrock RN, Ekramoddoullah AKM, Liu J-J, Yu X (2004) Gene cloning and tissue expression analysis of a PR-5 thaumatin-like protein in Phellinus weirii infected Douglas-fir. Phytopathology 94:1235–1243

  63. Zareie R, Melanson DL, Murphy PJ (2002) Isolation of fungal cell wall degrading proteins from barley (Hordeum vulgare L.) leaves infected with Rhynchosporium secalis. Mol Plant Microbe Interact 15:1031–1039

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This research was supported in part by Canadian Forest Service and CFS-Genomics R&D Initiative.

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Correspondence to Jun-Jun Liu.

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Liu, J., Zamani, A. & Ekramoddoullah, A.K.M. Expression profiling of a complex thaumatin-like protein family in western white pine. Planta 231, 637–651 (2010). https://doi.org/10.1007/s00425-009-1068-2

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  • Cronartium
  • Quantitative PCR
  • Phylogenetic analysis
  • Thaumatin-like protein
  • Western white pine