Probiotics and Antimicrobial Proteins

, Volume 11, Issue 1, pp 299–309 | Cite as

Gene Cloning, Expression, and Antifungal Activities of Permatin from Naked Oat (Avena nuda)

  • Jian Liu
  • Deping Han
  • Yawei ShiEmail author


Thaumatin-like proteins (TLPs) are the products of a large, highly complex gene family involved in host defense. TLPs also belong to the pathogenesis-related family 5 (PR-5) of plant defense proteins. Most TLPs exhibit potential antifungal activities, and their accumulation in the plant is related to many physiological processes. In this study, a gene encoding TLP named permatin with an open reading frame of 678 bp encoding a protein of 225 amino acids with a calculated molecular mass of 23.5 kDa was cloned from naked oat leaves. Phylogenetic analysis revealed that permatin shares high homology with a number of other TLPs among diverse taxa. Model of structure by homology modeling showed that permatin consists of an acidic cleft region consistent with most TLPs. Recombinant NusA-permatin was overexpressed in Escherichia coli strain BL21 and purified by Heparin column combined with Sephacryl S-200 column. The protein exhibited antifungal activity to Fusarium oxysporum (half maximal inhibitory concentration, IC50 = 21.42 μM). Morphological observation showed that NusA-permatin can induce mycelium deformation of F. oxysporum, the cell membrane is blurred, and the diaphragm is not obvious. NusA-permatin also causes membrane permeabilization and reactive oxygen species accumulation in the mycelium of F. oxysporum. Permatin may play an important role in the disease resistance responses of plants against pathogen attacks through its antifungal activity.


Permatin Fusarium oxysporum Gene cloning Recombinant expression Antifungal activity Naked oat 


Funding Information

We acknowledge that this project is supported by Key Research and Development Program of Shanxi Province of China (no.201603D211104), Special Funds of the Natural Science Foundation of TaiYuan, Shanxi Province (grant no. 11014908), and the fund for Shanxi “1331 project” collaborative innovation center and Research Project supported by Shanxi Scholarship Council of China (no.2017019).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Hakim, Ullah A, Hussain A, Shaban M, Khan AH, Alariqi M, Gul S, Jun Z, Lin S, Li J, Jin S, Munis M (2017) Osmotin: a plant defense tool against biotic and abiotic stresses. Plant Physiol Biochem 123:149–159CrossRefGoogle Scholar
  2. 2.
    van Loon LC, Rep M, Pieterse CM (2006) Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 44:135–162CrossRefGoogle Scholar
  3. 3.
    Finkina EI, Melnikova DN, Bogdanov IV, Ovchinnikova TV (2017) Plant pathogenesis-related proteins PR-10 and PR-14 as components of innate immunity system and ubiquitous allergens. Curr Med Chem 24:1772–1787CrossRefGoogle Scholar
  4. 4.
    Musidlak O, Nawrot R, Gozdzicka-Jozefiak A (2017) Which plant proteins are involved in antiviral defense? Review on in vivo and in vitro activities of selected plant proteins against viruses. Int J Mol Sci 18(11):1–23Google Scholar
  5. 5.
    Liu JJ, Sturrock R, Ekramoddoullah AK (2010) The superfamily of thaumatin-like proteins: its origin, evolution, and expression towards biological function. Plant Cell Rep 29:419–436CrossRefGoogle Scholar
  6. 6.
    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 27:1649–1659CrossRefGoogle Scholar
  7. 7.
    Smole U, Bublin M, Radauer C, Ebner C, Breiteneder H (2008) Mal d 2, the thaumatin-like allergen from apple, is highly resistant to gastrointestinal digestion and thermal processing. Int Arch Allergy Immunol 147:289–298CrossRefGoogle Scholar
  8. 8.
    Min K, Ha SC, Hasegawa PM, Bressan RA, Yun DJ, Kim KK (2004) Crystal structure of osmotin, a plant antifungal protein. Proteins 54:170–173CrossRefGoogle Scholar
  9. 9.
    Dall'Antonia Y, Pavkov T, Fuchs H, Breiteneder H, Keller W (2005) Crystallization and preliminary structure determination of the plant food allergen Pru av 2. Acta Crystallogr Sect F Struct Biol Cryst Commun 61:186–188CrossRefGoogle Scholar
  10. 10.
    Leone P, Menu-Bouaouiche L, Peumans WJ, Payan F, Barre A, Roussel A, Van Damme EJ, Rouge P (2006) Resolution of the structure of the allergenic and antifungal banana fruit thaumatin-like protein at 1.7-A. Biochimie 88:45–52CrossRefGoogle Scholar
  11. 11.
    Ghosh R, Chakrabarti C (2008) Crystal structure analysis of NP24-I: a thaumatin-like protein. Planta 228:883–890CrossRefGoogle Scholar
  12. 12.
    Skadsen RW, Sathish P, Kaeppler HF (2000) Expression of thaumatin-like permatin PR-5 genes switches from the ovary wall to the aleurone in developing barley and oat seeds. Plant Sci 156:11–22CrossRefGoogle Scholar
  13. 13.
    Ferreira RB, Monteiro S, Freitas R, Santos CN, Chen Z, Batista LM, Duarte J, Borges A, Teixeira AR (2007) The role of plant defence proteins in fungal pathogenesis. Mol Plant Pathol 8:677–700CrossRefGoogle Scholar
  14. 14.
    Ho BK, Thomas A, Brasseur R (2003) Revisiting the Ramachandran plot: hard-sphere repulsion, electrostatics, and H-bonding in the alpha-helix. Protein Sci 12:2508–2522CrossRefGoogle Scholar
  15. 15.
    Yan X, Qiao H, Zhang X, Guo C, Wang M, Wang Y, Wang X (2017) Analysis of the grape (Vitis vinifera L.) thaumatin-like protein (TLP) gene family and demonstration that TLP29 contributes to disease resistance. Sci Rep 7:4269CrossRefGoogle Scholar
  16. 16.
    Lin KC, Bushnell WR, Szabo LJ, Smith AG (1996) Isolation and expression of a host response gene family encoding thaumatin-like proteins in incompatible oat-stem rust fungus interactions. Mol Plant-Microbe Interact 9:511–522CrossRefGoogle Scholar
  17. 17.
    Wan Q, Hongbo S, Zhaolong X, Jia L, Dayong Z, Yihong H (2017) Salinity tolerance mechanism of osmotin and osmotin-like proteins: a promising candidate for enhancing plant salt tolerance. Curr Genomics 18:553–556CrossRefGoogle Scholar
  18. 18.
    Breiteneder H, Ebner C (2000) Molecular and biochemical classification of plant-derived food allergens. J Allergy Clin Immunol 106:27–36CrossRefGoogle Scholar
  19. 19.
    Yasmin N, Saleem M (2014) Biochemical characterization of fruit-specific pathogenesis-related antifungal protein from basrai banana. Microbiol Res 169:369–377CrossRefGoogle Scholar
  20. 20.
    Wang Q, Li F, Zhang X, Zhang Y, Hou Y, Zhang S, Wu Z (2011) Purification and characterization of a CkTLP protein from Cynanchum komarovii seeds that confers antifungal activity. PLoS One 6:e16930CrossRefGoogle Scholar
  21. 21.
    Chu KT, Ng TB (2003) Isolation of a large thaumatin-like antifungal protein from seeds of the Kweilin chestnut Castanopsis chinensis. Biochem Biophys Res Commun 301:364–370CrossRefGoogle Scholar
  22. 22.
    Acharya K, Pal AK, Gulati A, Kumar S, Singh AK, Ahuja PS (2013) Overexpression of Camellia sinensis thaumatin-like protein, CsTLP in potato confers enhanced resistance to Macrophomina phaseolina and Phytophthora infestans infection. Mol Biotechnol 54:609–622CrossRefGoogle Scholar
  23. 23.
    Jayaraj J, Punja ZK (2007) Combined expression of chitinase and lipid transfer protein genes in transgenic carrot plants enhances resistance to foliar fungal pathogens. Plant Cell Rep 26:1539–1546CrossRefGoogle Scholar
  24. 24.
    Sav H, Rafati H, Oz Y, Dalyan-Cilo B, Ener B, Mohammadi F, Ilkit M, van Diepeningen AD, Seyedmousavi S (2018) Biofilm formation and resistance to fungicides in clinically relevant members of the fungal genus fusarium. J Fungi (Basel) 4:1–12Google Scholar
  25. 25.
    Treikale O, Javoisha B, Feodorova-Fedotova L (2015) Occurrence of Fusarium species on small cereals in Latvia. Commun Agric Appl Biol Sci 80:551–554Google Scholar
  26. 26.
    Li Y, Mao L, Yan D (2014) Quantification of Fusarium oxysporum in fumigated soils by a newly developed real-time PCR assay to assess the efficacy of fumigants for Fusarium wilt disease in strawberry plants. Pest Manag Sci 70:1669–1675Google Scholar
  27. 27.
    Bellato S, Del FV, Redaelli R, Sgrulletta D, Bucci R, Magri AD, Marini F (2011) Use of near infrared reflectance and transmittance coupled to robust calibration for the evaluation of nutritional value in naked oats. J Agric Food Chem 59:4349–4360CrossRefGoogle Scholar
  28. 28.
    Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112:531–552Google Scholar
  29. 29.
    Jing L, Guo D, Hu W, Niu X (2017) The prediction of a pathogenesis-related secretome of Puccinia helianthi through high-throughput transcriptome analysis. BMC Bioinf 18:166CrossRefGoogle Scholar
  30. 30.
    Ashok KH, Venkatesh YP (2014) In silico analyses of structural and allergenicity features of sapodilla (Manilkara zapota) acidic thaumatin-like protein in comparison with allergenic plant TLPs. Mol Immunol 57:119–128CrossRefGoogle Scholar
  31. 31.
    Kim S, Lee J, Jo S, Brooks CR, Lee HS, Im W (2017) CHARMM-GUI ligand reader and modeler for CHARMM force field generation of small molecules. J Comput Chem 38:1879–1886CrossRefGoogle Scholar
  32. 32.
    Abdelmoteleb A, Troncoso-Rojas R, Gonzalez-Soto T, Gonzalez-Mendoza D (2017) Antifungical activity of autochthonous Bacillus subtilis isolated from Prosopis juliflora against phytopathogenic Fungi. Mycobiology 45:385–391CrossRefGoogle Scholar
  33. 33.
    Ma D, Li G, Zhu Y, Xie DY (2017) Overexpression and suppression of Artemisia annua 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1 gene (AaHDR1) differentially regulate artemisinin and terpenoid biosynthesis. Front Plant Sci 8:77Google Scholar
  34. 34.
    Guler-Gane G, Kidd S, Sridharan S, Vaughan TJ, Wilkinson TC, Tigue NJ (2016) Overcoming the refractory expression of secreted recombinant proteins in mammalian cells through modification of the signal peptide and adjacent amino acids. PLoS One 11:e0155340Google Scholar
  35. 35.
    Jimenez-Lopez JC, Robles-Bolivar P, Lopez-Valverde FJ, Lima-Cabello E, Kotchoni SO, Alche JD (2016) Ole e 13 is the unique food allergen in olive: structure-functional, substrates docking, and molecular allergenicity comparative analysis. J Mol Graph Model 66:26–40CrossRefGoogle Scholar
  36. 36.
    Ho VS, Wong JH, Ng TB (2007) A thaumatin-like antifungal protein from the emperor banana. Peptides 28:760–766CrossRefGoogle Scholar
  37. 37.
    Shi Y, Jian L, Han D, Ren Y (2015) Isolation of an antifungal pathogenesis-related protein from naked oat (Avena nuda) seeds. Cereal Chem 92:44–49CrossRefGoogle Scholar
  38. 38.
    Carmeille R, Croissant C, Bouvet F, Bouter A (2017) Membrane repair assay for human skeletal muscle cells. Methods Mol Biol 1668:195–207CrossRefGoogle Scholar
  39. 39.
    Dananjaya S, Udayangani R, Shin SY, Edussuriya M, Nikapitiya C, Lee J, De Zoysa M (2017) In vitro and in vivo antifungal efficacy of plant based laws one against Fusarium oxysporum species complex. Microbiol Res 201:21–29CrossRefGoogle Scholar
  40. 40.
    Correa A, Oppezzo P (2011) Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening. Biotechnol J 6:715–730CrossRefGoogle Scholar
  41. 41.
    Kumar S, Singh N, Sinha M, Kaur P, Srinivasan A, Sharma S, Singh TP (2009) Isolation, purification, crystallization and preliminary crystallographic studies of amaryllin, a plant pathogenesis-related protein from Amaryllis belladonna. Acta Crystallogr Sect F Struct Biol Cryst Commun 65:635–637CrossRefGoogle Scholar
  42. 42.
    Jami SK, Swathi AT, Guruprasad L, Kirti PB (2007) Molecular, biochemical and structural characterization of osmotin-like protein from black nightshade (Solanum nigrum). J Plant Physiol 164:238–252CrossRefGoogle Scholar
  43. 43.
    Ramos MV, de Oliveira RS, Pereira HM, Moreno FB, Lobo MD, Rebelo LM, Brandao-Neto J, de Sousa JS, Monteiro-Moreira AC, Freitas CD, Grangeiro TB (2015) Crystal structure of an antifungal osmotin-like protein from Calotropis procera and its effects on Fusarium solani spores, as revealed by atomic force microscopy: insights into the mechanism of action. Phytochemistry 119:5–18CrossRefGoogle Scholar
  44. 44.
    Perri F, Della PS, Rufini F, Patamia M, Bonito M, Angiolella L, Vitali A (2009) Antifungal-protein production in maize (Zea mays) suspension cultures. Biotechnol Appl Biochem 52:273–281CrossRefGoogle Scholar
  45. 45.
    de Freitas CD, Lopes JL, Beltramini LM, de Oliveira RS, Oliveira JT, Ramos MV (2011) Osmotin from Calotropis procera latex: new insights into structure and antifungal properties. Biochim Biophys Acta 1808:2501–2507CrossRefGoogle Scholar
  46. 46.
    de Freitas CD, Nogueira FC, Vasconcelos IM, Oliveira JT, Domont GB, Ramos MV (2011) Osmotin purified from the latex of Calotropis procera: biochemical characterization, biological activity and role in plant defense. Plant Physiol Biochem 49:738–743CrossRefGoogle Scholar
  47. 47.
    Souza I, Ramos MV, Costa JH, Freitas C, Oliveira R, Moreno FB, Moreira RA, Carvalho C (2017) The osmotin of Calotropis procera latex is not expressed in laticifer-free cultivated callus and under salt stress. Plant Physiol Biochem 119:312–318CrossRefGoogle Scholar
  48. 48.
    Elhouiti F, Tahri D, Takhi D, Ouinten M, Barreau C, Verdal-Bonnin MN, Bombarda I, Yousfi M (2017) Variability of composition and effects of essential oils from Rhanterium adpressum Coss. & Durieu against mycotoxinogenic Fusarium strains. Arch Microbiol 199:1345–1356CrossRefGoogle Scholar
  49. 49.
    Bleackley MR, Wiltshire JL, Perrine-Walker F, Vasa S, Burns RL, van der Weerden NL, Anderson MA (2014) Agp2p, the plasma membrane transregulator of polyamine uptake, regulates the antifungal activities of the plant defensin NaD1 and other cationic peptides. Antimicrob Agents Chemother 58:2688–2698CrossRefGoogle Scholar
  50. 50.
    van der Weerden NL, Bleackley MR, Anderson MA (2013) Properties and mechanisms of action of naturally occurring antifungal peptides. Cell Mol Life Sci 70:3545–3570CrossRefGoogle Scholar
  51. 51.
    Palacin A, Tordesillas L, Gamboa P, Sanchez-Monge R, Cuesta-Herranz J, Sanz ML, Barber D, Salcedo G, Diaz-Perales A (2010) Characterization of peach thaumatin-like proteins and their identification as major peach allergens. Clin Exp Allergy 40:1422–1430CrossRefGoogle Scholar
  52. 52.
    Villa NY, Moussatche P, Chamberlin SG, Kumar A, Lyons TJ (2011) Phylogenetic and preliminary phenotypic analysis of yeast PAQR receptors: potential antifungal targets. J Mol Evol 73:134–152CrossRefGoogle Scholar
  53. 53.
    Aimanianda V, Simenel C, Garnaud C, Clavaud C, Tada R, Barbin L, Mouyna I, Heddergott C, Popolo L, Ohya Y, Delepierre M, Latge JP (2017) The dual activity responsible for the elongation and branching of beta-(1,3)-glucan in the fungal cell wall. MBio 8:e00619–e00617CrossRefGoogle Scholar
  54. 54.
    Ibeas JI, Lee H, Damsz B, Prasad DT, Pardo JM, Hasegawa PM, Bressan RA, Narasimhan ML (2000) Fungal cell wall phosphomannans facilitate the toxic activity of a plant PR-5 protein. Plant J 23:375–383CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of EducationShanxi UniversityTaiyuanPeople’s Republic of China

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