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Obtaining Transgenic Potato Plants Expressing the Human Lactoferrin Gene and Analysis of Their Resistance to Phytopathogens

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Abstract

The human lactoferrin gene was transferred into genomes of several potato (Solanum tuberosum) cultivars of Ukrainian breeding using the Agrobacterium-mediated transformation. The plasmid vector pBIN35LF carrying the human lactoferrin gene hLf controlled by the 35S cauliflower mosaic virus promoter (CaMV35S) and the octopine synthase terminator, as well as the selective marker neomycin phosphotransferase II gene (nptII) conferring the resistance to kanamycin, was used. As a result of selection, 44 lines of Vernisage, 26 lines of cv. Levada, 25 lines of cv. Svitanok Kyivskyi, and 16 lines of cv. Zarevo cultivars resistant to 100 mg/L of kanamycin were obtained. PCR and Western blot analyses were carried out for transformed lines with primers specific to the hLf gene and a monoclonal antibody against lactoferrin to confirm the transgenic nature of selected tomato plants and hLf gene expression. The selected transgenic potato lines were tested for resistance to bacterial and fungal phytopathogens. The disk diffusion assay revealed that the juice of transgenic potato lines possesses an antibacterial effect against phytopathogenic bacteria Ralstonia solanacearum (causing potato brown rot) and Clavibacter michiganensis subsp. sepedonicus (causing potato ring rot). The resistance of transgenic potato plants to late blight was investigated by in vitro infection of plants with the Phytophthora infestans isolate. As a result, it was found that the obtained transgenic potato lines have enhanced resistance to P. infestans as compared to the control. Thus, the obtained data show that the transfer of the hLf gene into the potato genome enhances potato’s resistance to bacterial and fungal pathogens.

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REFERENCES

  1. Melnyk, S.I., Kovchi, A.L., Stefkivska, Y.L., Kravchuk, O.O., and Gorytska, T.V., Potato market in Ukraine, Plant Var. Stud. Protect., 2017, vol. 13, no. 2, pp. 206–210. https://doi.org/10.21498/2518-1017.13.2.2017.105419

    Article  Google Scholar 

  2. Cook, E., Agriculture, Forestry and Fishery Statistics, Luxembourg: Publications Office of the European Union, 2018.

    Google Scholar 

  3. Kolobayev, V.A. and Rogozina, Ye.V., Cultivars immunity to phytopathogens as a factor of environmental safety: the cases of potato and sugarcane, Biosphere, 2014, vol. 6, no. 3, pp. 222–230.

    Google Scholar 

  4. Erenkova, L.A., Molyavko, A.A., Marukhlenko, A.V., and Borisova, N.P., New generation potato varieties with resistance to pathogens, Breeding, Seed Product.Genet., 2018, vol. 4, pp. 47–50. https://doi.org/10.24411/2413-4112-2018-10007

    Article  Google Scholar 

  5. Fiers, M., Edel-Hermann, V., Chatot, C., Alabouvette, C., and Steinberg, C., Potato soil-borne diseases. A review, Agron. Sustainable Dev., 2012, vol. 32, pp. 93–132. https://doi.org/10.1007/s13593-011-0035-z

    Article  Google Scholar 

  6. Gordienko, V.V. and Zaharchuk, N.A., Creation of initial breeding material of potato with complex resistance to Fusarium dry rot and tuber late blight, Plant Varieties Stud. Protect., 2017, vol. 13, no. 3, pp. 239–244.https://doi.org/10.21498/2518-1017.13.3.2017

  7. Kolomiets, Y.V., Grygoryuk, I.P., and Butsenko, L.M., Bacterial diseases of tomato plants in terms of open and covered growing of Ukraine, Ann. Agrar. Sci., 2017, vol. 15, no. 2, pp. 213–216. https://doi.org/10.1016/j.aasci.2017.05.010

    Article  Google Scholar 

  8. Kolomiets, Y.V., Grygoryuk, I.P., Butsenko, L.M., and Kalinichenko, A.V., Biotechnological control methods against phytopathogenic bacteria in tomatoes, Appl. Ecol. Environ. Res., 2019, vol. 17, no. 2, pp. 3215– 3230. https://doi.org/10.15666/aeer/1702_32153230

    Article  Google Scholar 

  9. Kolomiets, Y.V., Grygoryuk, I.P., Likhanov A., But-senko L.M., and Blume Ya., Induction of bacterial canker resistance in tomato plants using plant growth promoting Rhizobacteria, Open Agric. J., 2019, vol. 13, pp. 215–22.https://doi.org/10.2174/18743315019

  10. Boroday, V.V. and Parfeniuk, A.I., Spreading and development of the main potato (Solanum tuberosum L.) diseases in Ukraine, Agroecol. J., 2018, vol. 4, pp. 82–87. https://doi.org/10.33730/2077-4893.4.2018.161774

    Article  Google Scholar 

  11. Lopez-Garcia, B., San Segundo, B., and Coca, M., Antimicrobial peptides as a promising alternative for plant disease protection, in Small Wonders:Peptides for Disease Control, 2012, pp. 263–294. https://doi.org/10.1021/bk-2012-1095.ch013

    Google Scholar 

  12. Moosa, A., Farzand, A., Sahi, S.T., and Khan, S.A., Transgenic expression of antifungal pathogenesis-related proteins against phytopathogenic fungi—15 years of success, Israel. J. Plant Sci., 2017. doi 80/07929978.2017.1288407

  13. Jung, Y., Kang, K., Application of antimicrobial peptides for disease control in plants, Plant Breed. Biotechnol., 2014, vol. 2, no. 1, pp. 1–13. https://doi.org/10.9787/PBB.2014.2.1.001

    Article  Google Scholar 

  14. Patil, V.U., Gopal, J., and Singh, B.P., Improvement for bacterial wilt resistance in potato by conventional and biotechnological approaches, Agric. Res., 2012, vol. 1, pp. 299–316. https://doi.org/10.1007/S40003-012-0034-6

    Article  CAS  Google Scholar 

  15. Yemets, A.I., Tanasienko, I.V., Krasylenko, Y.A., and Blume, Y.B., Plant-based biopharming of recombinant human lactoferrin, Cell Biol. Int., 2014, vol. 38, pp. 989–1002. https://doi.org/10.1002/cbin.10304

    Article  CAS  PubMed  Google Scholar 

  16. Murashige, T. and Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant.,1962, vol. 15, pp. 473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

    Article  CAS  Google Scholar 

  17. Buziashvili, A. and Yemets, A., Obtaining of tomato and potato plants with human lactoferrin gene hLf, Rep. Natl. Acad. Sci. Ukr., 2018, vol. 10, pp. 88–94. https://doi.org/10.15407/dopovidi2018.10.088

    Article  CAS  Google Scholar 

  18. Sambrook, J.F. and Russell, D.W., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 2001.

    Google Scholar 

  19. Jeroen, S., Damm, B., Melchers, L., and Hoekema, A., Factors influencing transformation frequency of tomato (Lycopersicon esculentum), Plant Cell Rep., 1993, vol. 12, pp. 644–647. https://doi.org/10.1007/BF00232816

    Article  Google Scholar 

  20. Rogers, S.O., Bendich, A.J., Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues, Plant Mol. Biol., 1985, vol. 5, pp. 69–76. https://doi.org/10.1007/BF00020088

    Article  CAS  PubMed  Google Scholar 

  21. Tanasienko, I.V., Yemets, A.I., Pirko, Y.V., Korhkovyy, V.I., Abumhadi, N., and Blume, Y.B., Generation of transgenic barley lines producing human lactoferrin using mutant alpha-tubulin gene as the selective marker, Cytol. Genet., 2011, vol. 45, no. 1, pp. 3–10. https://doi.org/10.3103/S0095452711010026

    Article  CAS  Google Scholar 

  22. Baskaran, P., Soys, V., Balazs, E., and Van Stadena, J., Shoot apical meristem injection: A novel and efficient method to obtain transformed cucumber plants, S. Afr. J. Bot., 2016, vol. 103, pp. 210–215. https://doi.org/10.1016/j.sajb.2015.09.006

    Article  CAS  Google Scholar 

  23. Mitra, A., Zhang, Z., Expression of a human lactoferrin cDNA in tobacco cells produces antibacterial protein(s), Plant Physiol., 1994, vol. 106, no. 3, pp. 977–981.

    Article  CAS  Google Scholar 

  24. Rachmawati, D., Mori, T., Hosaka, T., Takaiwa, F., Inoue, E., and Anzai, H., Production and characterization of recombinant human lactoferrin in transgenic Javanica rice, Breed. Sci., 2005, vol. 55, no. 2, pp. 213–222. https://doi.org/10.1270/jsbbs.55.213

    Article  CAS  Google Scholar 

  25. Bradford, M., A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 1976, vol. 72, no. 1–2, pp. 248–254.

    Article  CAS  Google Scholar 

  26. Laemmli, U., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, vol. 227, pp. 680–5.

    Article  CAS  Google Scholar 

  27. Onuoha, S.C., Alisa, C.O., Antimicrobial potential of leaf juice and extracts of Moringa oleifera lam against some human pathogenic bacteria, J. Pharm. Biol. Sci., 2013, vol. 5, no. 4, pp. 37–42. https://doi.org/10.9790/3008-0543742

    Article  CAS  Google Scholar 

  28. Chahardooli, M., Farzaneh, A., and Sohrabi, A., Expression of recombinant Arabian camel lactofer-ricin-related peptide in Pichia pastoris and its antimicrobial identification, J. Sci. Food Agricult., 2016, vol. 96, no. 2, pp. 569–575. https://doi.org/10.1002/jsfa.7125

    Article  Google Scholar 

  29. Yabuuchi, E., Kosako, Y., Oyaizu, H., Yano, I., Hotta, H., Hashimoto, Y., Ezaki, T., and Arakawa, M., Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov., Microbiol. Immunol., 1992, vol. 36, no. 12, pp. 1251–75. https://doi.org/10.1111/j.1348-0421.1992.tb02129.x

    Article  CAS  PubMed  Google Scholar 

  30. Podgorskiy, V.S., Ukrainian National Collection of Microorganisms, 2nd ed., Kyiv: Naukova Dumka, 2007.

    Google Scholar 

  31. Tournas, V., Stack, M.E., Mislivec, P.B., Koch, H.A., and Bandler, R., FDA Bacteriological Analytical Manual, chapter 18: Yeasts, Molds, and Mycotoxins, 8th ed., Revision A, USA: AOAC International, 1998.

  32. Tkachyk, S.O., Methods of Phytopathological Researches with Artificial Infection of Plants, Kyiv: Nylan-LTD, 2014.

    Google Scholar 

  33. Cherednichenko, L.M., Evaluation of the potato selection material for the resistance of the aboveground plant parts to late blight with the use of detached leaf particles, Potato Grow. Ukr., 2013, vols. 1–2, pp. 19–24.

    Google Scholar 

  34. Podhaietskyi, A.A., Kravchenko, N.V., Kovalenko, V.M., Bondus, R.O., Hordienko, V.V., Cherednichenko, L.M., and Sobra, V.M., Ecological testing of potatoes, Ukr. J. Ecol., 2018, vol. 8, no. 4, pp. 17–25.

    Article  Google Scholar 

  35. Michalska, A.M., Sobkowiak, S., Flis, B. Zimnoch-Guzowska, E., Virulence and aggressiveness of Phytophthora infestans isolates collected in Poland from potato and tomato plants identified no strong specificity, Eur. J. Plant Pathol., 2016, vol. 144, pp. 325–336. https://doi.org/10.1007/s10658-015-0769-6

    Article  Google Scholar 

  36. Buziashvili A.Yu. and Yemets, A.I., Agrobacterium-mediated transformation of potato with human lactoferrin gene, Fact. Exp. Evol. Org., 2019, vol. 25, pp. 184–189.

    Google Scholar 

  37. Han, E.H., Goo, Y.M., Lee, M.K., and Lee, S.W., An efficient transformation method for a potato (Solanum tuberosum L. var. Atlantic), J. Plant Biotechnol., 2015, vol. 42, pp. 77–82. https://doi.org/10.5010/JPB.2015.42.2.77

    Article  Google Scholar 

  38. Shin, D.Y., Seong, E.S., Na, J.K., Yoo, J.H., Kang, W.H., Ghimire, B.K., Lim, J.D., Yu, C.Y., Conditions for regeneration and transformation of Solanum tuberosum cultivars using the cotton glutathione S-transferase (Gh-5) gene, Afr. J. Biotechnol., 2011, vol. 10, no. 67, pp. 15135–15141. https://doi.org/10.5897/AJB11.1895

    Article  CAS  Google Scholar 

  39. Soto, N., Enriquez, G.A., Ferreira, A., Corrada, M., Fuentes, A., Tiel, K., and Pujol, M., Efficient transformation of potato stems segments from cultivar Desiree, using phosphinothricin as selection marker, Biotechnol. Apl., 2007, vol. 24, no. 2, pp. 139–144.

    Google Scholar 

  40. Craze, M., Bates, R., Bowden, S., and Wallington, E.J., Highly efficient Agrobacterium-mediated transformation of potato (Solanum tuberosum) and production of transgenic microtubers, Curr. Protoc. Plant Biol., 2018, vol. 3, no. 1, pp. 33–41. doi 10.1002/cppb.20065

  41. Alimohammadi, M., Bagherieh-Najjar, M., Agrobacterium-mediated transformation of plants: basic principles and influencing factors, Afr. J. Biotechnol., 2009, vol. 8, no. 20, pp. 5142–5148.

    CAS  Google Scholar 

  42. Opabode, J., Agrobacterium-mediated transformation of plants: emerging factors that influence efficiency, Biotechnol. Mol. Biol. Rev., 2006, vol. 1, no. 1, pp. 12–20.

    Google Scholar 

  43. Bakhsh, A., Anayol, E., and Ozcan, S., Comparison of transformation efficiency of five Agrobacterium tumefaciens strains in Nicotiana tabacum L., Emirates J. Food Agricult., 2014, vol. 26, no. 3, pp. 259–264. https://doi.org/10.9755/ejfa.v26i3.16437

    Article  Google Scholar 

  44. Gelvin, S., Liu, C., Genetic manipulation of Agrobac-terium tumefaciens strains to improve transformation of recalcitrant plant species, Plant Mol. Biol. Manual., 1994, pp. 85–97.

    Google Scholar 

  45. Stefanova, G., Slavov, S., Gecheff, K., Vlahova, M., and Atanassov, A., Expression of recombinant human lactoferrin in transgenic alfalfa plants, Biol. Plant., 2013, vol. 57, no. 3, pp. 457–464.

    Article  CAS  Google Scholar 

  46. Vlahova, M., Stefanova, G., Petkov, P., Barbulova, A., Petkova, D., Kalushkov, P., and Atanassov, A., Genetic modification of alfalfa (Medicago sativa L.) for quality improvement and production of novel compounds, Biotechnol. Biotechn. Equipm., 2005, vol. 19, no. 3, pp. 56–62. doi 0/13102818.2005.10817286

  47. Legrand, D., Salmon, V., Spik, G., Gruber, V., Bournat, P., and Merot, B., Recombinant lactoferrin, methods of production from plants and uses, US Patent No. 6569831 B1, May 27, 2003.

  48. Chong, D.K., Langridge, W.H., Expression of full-length bioactive antimicrobial human lactoferrin in potato plants, Transgen. Res., 2000, vol. 9, no. 1, pp. 71–78. https://doi.org/10.1023/A:1008977630179

    Article  CAS  Google Scholar 

  49. On the Amendments to the List of Regulated Pests, The edition of the order of the Ministry of Agrarian Policy and Food of Ukraine, July 16, 2019.

  50. Fukuta, S., Kawamoto, K., Mizukami, Y., Yoshimura, Y., Ueda, J., and Kanbe, M., Transgenic tobacco plants expressing antimicrobial peptide bovine lactoferricin show enhanced resistance to phyto-pathogens. Plant Biotechnol., 2012, vol. 29, no. 4, pp. 383–389. https://doi.org/10.5511/plantbiotechnology.12.0619a

    Article  CAS  Google Scholar 

  51. Sohrabi, S.M., Niazi, A., Chahardooli, M., and Aram, F., Isolation and expression of antimicrobial camel lactoferrin (cLf) gene in tobacco, Plant OMICS, 2014, vol. 7, pp. 298–307.

    CAS  Google Scholar 

  52. Chahardooli, M., Fazeli, A., Niazi, A., and Ghabooli, M., Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria, Biotechnol. Biotechn. Equipm., 2018, vol. 32, no 3, pp. 714–723. https://doi.org/10.1080/13102818.2018.1451780

    Article  CAS  Google Scholar 

  53. Humphrey, B.D., Huang, N., and Klasing, K.C., Rice expressing lactoferrin and lysozyme has antibiotic-like properties when fed to chicks, J. Pediatr. Gastroenterol. Nutr., 2003, vol. 36, no. 2, pp. 190–199. https://doi.org/10.1093/jn/132.6.1214

    Article  Google Scholar 

  54. Lee, T.T., Chang, C.C., Juang, R.S., Chen, R.B., Yang, H.Y., Chu, L.W., Wang, S.R., Tseng, T.H., Wang, C.S., Chen, L.J., and Bi, Yu., Porcine lactoferrin expression in transgenic rice and its effects as a feed additive on early weaned piglets, J. Agric. Food Chem., 2010, vol. 58, no. 8, pp. 5166–5173. https://doi.org/10.1021/jf903904s

    Article  CAS  PubMed  Google Scholar 

  55. Takase, K., Hagiwara, K., Onodera, H., Nishizawa, Y., Ugaki, M., Omura, T., Numata, S., Akutsu, K., Kumura, H., and Shimazaki, K., Constitutive expression of human lactoferrin and its N-lobe in rice plants to confer disease resistance, Biochem. Cell Biol., 2005, vol. 83, no. 2, pp. 239–49. https://doi.org/10.1139/o05-022

    Article  CAS  PubMed  Google Scholar 

  56. Franco, I., Castillo, E., Perez, M.D., Calvo, M., and Sanchez, L., Effects of hydrostatic high pressure on the structure and antibacterial activity of recombinant human lactoferrin from transgenic rice, Biosci. Biotechnol. Biochem., 2012, vol. 76, no. 1, pp. 53–59. https://doi.org/10.1271/bbb.110433

    Article  CAS  PubMed  Google Scholar 

  57. Funakoshi, T., Hosaka, T., Inoue, E., and Anzai, H., Gene expression of lactoferrin-derived antimicrobial peptides in rice, Plant Physiol. Biochem., 2017, vol. 123. https://doi.org/10.1016/j.plaphy.2017.12.037

  58. Lee, T. J., Coyne, D. P., Clemente, T. E., and Mitra, A., Partial resistance to bacterial wilt in transgenic tomato plants expressing antibacterial lactoferrin gene, J. Am. Soc. Horticult. Sci., 2002, vol. 127, no. 2, pp. 158–164. https://doi.org/10.21273/JASHS.127.2.158

    Article  CAS  Google Scholar 

  59. Jo, S.H., Kwon, S.Y., Park, D.S., Yang, K.S., Kim, J.W., Lee, K.T., Kwak, S.S., and Lee, H.S., High-yield production of functional human lactoferrin in transgenic cell cultures of Siberian ginseng (Acanthopanax senticosus), Biotechnol. Bioprocess Eng., 2006, vol. 11, no. 5, pp. 442–448. https://doi.org/10.1007/BF02932312

    Article  CAS  Google Scholar 

  60. Malnoy, M., Venisse, J.S., Brisset, M.N., and Chevreau, E., Expression of bovine lactoferrin cDNA confers resistance to Erwinia amylovora in transgenic pear, Mol. Breed., 2003, vol. 12, no. 3, pp. 231–244. https://doi.org/10.1023/A:1026365311067

    Article  CAS  Google Scholar 

  61. Nguyen, T.C., Lakshman, D.K., Han, J., Galvez, L.C., and Mitra, A., Transgenic plants expressing antimicrobial lactoferrin protein are resistant to a fungal pathogen, J. Plant Mol. Biol. Biotechnol., 2011, vol. 2, no.1, pp. 1–8.

    CAS  Google Scholar 

  62. Han, J., Lakshman, D.K., Galvez, L.C., Mitra, S., Baenziger, P.S., and Mitra, A., Transgenic expression of lactoferrin impacts enhanced resistance to head blight of wheat caused by Fusarium graminearum, BMC Plant Biol., 2012, vol. 12, no. 33. https://doi.org/10.1186/1471-2229-12-33

  63. Bruni, N., Capucchio, M.T., Biasibetti, E., Pessione, E., Cirrincione, S., Giraudo, L., Corona, A., and Dosio, F., Antimicrobial activity of lactoferrin-related peptides and applications in human and veterinary medicine, Molecules, 2016, vol. 21, no. 6. https://doi.org/10.3390/molecules21060752

  64. Fernandes, K.E., Carter, D.A., The antifungal activity of lactoferrin and its derived peptides: mechanisms of action and synergy with drugs against fungal pathogens, Front. Microbiol., 2017, vol. 8, no. 2. https://doi.org/10.3389/fmicb.2017.00002

  65. Fry, W., Phytophthora infestans: the plant (and R gene) destroyer, Mol. Plant Pathol., 2008, vol. 9, no. 3, pp. 385–402.https://doi.org/10.1111/j.1364-3703.2007

  66. Dominguez, P., Gomez, I., Barbero, M., Fellmann, T., Chatzopoulos, T., Jensen, H., and Philippidis, G., Eu Commodity Market Development: Medium-Term Agricultural Outlook, Luxembourg: Publ. Office. Eur. Union, 2018, ISBN 978-92-79-97850-0, https://doi.org/10.2760/4519, JRC113987

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Funding

This research was financially supported for AB, YB and AY by the grant “Application of lactoferrin gene for production of phytopathogen resistant plant lines from Solanaceaå family” in the frames of complex interdisciplinary research program “Molecular and Cell Biotechnologies for the Needs of Medicine, Industry, and Agriculture” of National Academy of Sciences of Ukraine (no. 0115U005021, 2015–2019).

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Authors and Affiliations

  1. Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine, 04123, Kyiv, Ukraine

    A. Buziashvili, Ya. B. Blume & A. Yemets

  2. Institute of Potato, National Academy of Agrarian Sciences of Ukraine, 07853, Kyiv, Ukraine

    L. Cherednichenko

  3. Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 03860, Kyiv, Ukraine

    S. Kropyvko

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  1. A. Buziashvili
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  2. L. Cherednichenko
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  4. Ya. B. Blume
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Correspondence to A. Buziashvili or A. Yemets.

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Buziashvili, A., Cherednichenko, L., Kropyvko, S. et al. Obtaining Transgenic Potato Plants Expressing the Human Lactoferrin Gene and Analysis of Their Resistance to Phytopathogens. Cytol. Genet. 54, 179–188 (2020). https://doi.org/10.3103/S0095452720030020

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