Interference inhibition of Plum pox virus, induced by a hairpin-RNA of viral origin, provides long-term resistance to PPV infection in adult plants of the Startovaya (Prunus domestica L.) variety

In modern horticulture Plum pox virus (PPV) imposes serious threats to commercial plantations of a wide range of fruit species belonging to genera Prunus. Given the lack of natural genetic resources, which display reliable resistance to PPV infection, there has been considerable interest in using genetic engineering methods for targeted genome modification of stone fruit trees to control Sharka disease caused by PPV. Among the many virus defense mechanisms, RNA interference is shown to be the most promising transgenic disease-control strategy in plant biotechnology. The present study describes the production of transgenic PPV resistant European plum `Startovaya` (P. domestica L.) through the Agrobacterium-mediated transformation of in vitro leaf explants. Due to organogenesis from leaves, the established protocol allows the genetic engineering of the plum genome without losing clonal fidelity of original cultivar. Seven independent transgenic plum lines containing the self-complementary fragments of PPV-CP gene sequence separated by a PDK intron were generated using hpt as a selective gene and uidA as a reporter gene. The transformation was verified through the histochemical staining for β-glucuronidase activity, PCR amplification of appropriate vector products from isolated genomic DNA and Southern blot analysis of hairpin PPV-CP gene fragments. To clarify the virus resistance, plum buds infected by PPV-M strain were grafted onto 1-year-old transgenic plants, which further were grown into mature trees in the greenhouse. As evaluated by RT-PCR, DAS-ELISA, Western blot, Immuno Strip test, and visual observations, GM plum trees remained uninfected over 9 years. Infected branches that developed from grafted buds displayed obvious symptoms of Sharka disease over the years and maintained the high level of virus accumulation, whereby host transgenic trees had been constantly challenged with the pathogen. Since the virus was unable to spread to transgenic tissues, the stable expression of PPV-derived gene

1. Scholthof K. B., Adkins S., Czosnek H., Palukaitis P. Jacquot E., Hohn T. et al. Top 10 plant viruses in molecular plant pathology. Mol. Plant Pathol. 2011;12:938-954. DOI:10.1111/j.1364-3703.2011.00752.x

2. Garcia J. A., Glasa M., Cambra M. and Candresse T. Plum pox virus and sharka: a model 436 potyvirus and a major disease. Mol. Plant Pathol. 2014;15:226-241. DOI:10.1111/mpp.12083

3. Rimbaud L., Dallot S., Gottwald T., Decroocq V., Jacquot E., Soubeyrand S. et al. Sharka epidemiology and worldwide management strategies: learning lessons to optimize disease control in perennial plants. Annu. Rev. Phytopathol. 2015;53:357-378. DOI:10.1146/annurev-phyto-080614-120140

4. Neumüller M. and Hartmann W. The phenotypically quantitative nature of hypersensitivity of European plum (Prunus domestica L.) against the Plum pox virus and its description using the hypersensitivity index. Hort. Sci. 2008;35:50-64. DOI:10.17221/639-HORTSCI

5. Scorza R., Ravelonandro M., Callahan A. M., Cordts J. M., Fuchs M., Dunez J. et al. Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene. Plant Cell Rep. 1994;14:18-22. DOI:10.1007/BF00233291

6. Ilardi V., and Tavazza M. Biotechnological strategies and tools for plum pox virus resistance: trans-, intra-, cis-genesis, and beyond. Front. Plant Sci. 2015;6:379. DOI:10.3389/fpls.2015.00379

7. Limera C., Sabbadini S., Sweet J. B. and Mezzetti B. New biotechnological tools for the genetic improvement of major woody fruit species. Front. Plant Sci. 2017;8:1418. DOI:10.3389/fpls.2017.0141

8. Kundu J. K., Briard P., Hily J. M., Ravelonandro M. and Scorza R. Role of the 25–26 nt siRNA in the resistance of transgenic Prunus domestica graft inoculated with plum pox virus. Virus Genes 2008;36:215-220. DOI:10.1007/s11262-007-0176-y

9. Capote N., Perez-Panades J., Monzo C., Carbonell E., Urbaneja A., Scorza R. et al. Assessment of the diversity and dynamics of Plum pox virus and aphid populations in transgenic European plums under Mediterranean conditions. Transgenic Res. 2008;17:367-377. DOI:10.1007/s11248-007-9112-0

10. Polák J., Ravelonandro M., Kundu J. K., Pívalová J., and Scorza R. Interactions of Plum Pox Virus strain Rec with Apple Chlorotic Leafspot Virus and Prune Dwarf Virus in fi eld-grown transgenic plum Prunus domestica L., clone C5. Plant Protect. Sci. 2008;44:1-5. DOI:10.17221/535-PPS

11. Polák J., Kundu J. K., Krška B., Beoni E., Komínek P., Pívalová J. et al. Transgenic plum Prunus domestica L., clone C5 (cv. HoneySweet) forprotection against sharka disease. J. Integr. Agric. 2017;16:516-522. DOI:10.1016/S2095-3119(16)61491-0

12. Wong W., Barba P., Álvarez C., Castro Á, Acun´a M., Zamora P. et al. Evaluation of the resistance of transgenic C5 plum (Prunus domestica l.) against four Chilean Plum pox virus isolates through micro-graft ing. Chil. J. Agric. Res. 2009;70:372-380.

13. Scorza R., Callahan A., Dardick C., Ravelonandro M., Polák J., Malinowski T. et al. Genetic engineering of Plum pox virus resistance: honey sweet plum from concept to product. Plant Cell Tiss. Organ. Cult. 2013;115:1-12. DOI:10.1007/s11240-013-0339-6

14. Khalid A., Zhang Q., Yasir M., and Li F. Small RNA based genetic engineering for plant viral resistance: application in crop protection. Front. Microbiol. 2017;8:43. DOI:10.3389/fmicb.2017.0004

15. Hily J. M., Ravelonandro M., Damsteegt V., Bassett C., Petri C., Liu Z. et al. Plum pox virus coat protein gene intron-hairpin- RNA (ihpRNA) constructs provide resistance to Plum pox virus in Nicotiana benthamiana and Prunus domestica. J. Am. Soc. Hort. Sci. 2007;132:850-858. DOI:10.21273/JASHS.132.6.850

16. Wang A., Tian L., Huang T. S., Brown D. C. W., Svircev A. M., Stobbs L. W. et al. The development of genetic resistance to Plum pox virus in transgenic Nicotiana benthamiana and Prunus domestica. Acta Hort. 2009;839:665-672. DOI:10.17660/ActaHortic.2009.839.91

17. Monticelli S., Di Nicola-Negri E., Gentile A., Damiano C. and Ilardi V. Production and in vitro assessment of transgenic plums for resistance to Plum pox virus: a feasible, environmental risk-free, cost-effective approach. Ann. Appl. Biol. 2012;161:293- 301. DOI:10.1111/j.1744-7348.2012.00573.x

18. Petri C., Hily J. M., Vann C., Dardick C. and Scorza R. A high-throughput transformation system allows the regeneration of marker-free plum plants (Prunus domestica). Ann. Appl. Biol. 2011;159:302-315. DOI:10.1111/j.1744-7348.2011.00499.x

19. Wang A. Tian L., Brown D. C. W., Svircev A. M., Stobbs L. W. and Sanfaçon H. Generation of effi cient resistance to Plum pox virus (PPV) in Nicotiana benthamiana and Prunus domestica expressing triple-intron-spanned double-hairpin RNAs simultaneously targeting 5’ and 3’ conserved genomic regions of PPV. Acta Hort. 2013;1063:77-84. DOI:10.17660/ActaHortic.2015.1063.9

20. García-Almodóvar R. C., Clemente-Moreno M. J., Díaz- Vivancos P., Petri C., Rubio M., Padilla I. M. G. et al. Greenhouse evaluation confirms in vitro sharka resistance of genetically engineered h-UTR/P1plum plants. Plant Cell Tiss. Organ. Cult. 2015;120:791-796. DOI:10.1007/s11240-014-0629-7

21. Decroocq V., Sicard O., Alamillo J. M., Lansac M., Eyquard J. P., García J. A. et al. Multiple resistance traits control Plum pox virus infection in Arabidopsis thaliana. Mol. Plant Microbe Interact. 2006;19:541-549. DOI:10.1094/MPMI-19-0541

22. Marandel G., Salava J., Abbott A., Candresse T. and Decroocq V. Quantitative trait loci meta-analysis of Plum pox virus resistance in apricot (Prunus armeniaca L.): new insights on the organization and the identification of genomic resistance factors. Mol. Plant Pathol. 2009;10:347-360. DOI:10.1111/j.1364-3703.2009.00535.x

23. Wang A. and Krishnaswamy S. Eukaryotic translation initiation factor 4E- mediated recessive resistance to plant viruses and its utility in crop improvement. Mol. Plant Pathol. 2012;13:795- 803. DOI:10.1111/J.1364-3703.2012.00791.X

24. Wang X., Kohalmi S. E., Svircev A., Wang A., Sanfacon H., and Tian L. Silencing of the host factor eIF(iso)4E gene confers plum pox virus resistance in plum. PLoS One. 2013;8:e50627. DOI:10.1371/journal.pone.0050627

25. Petri C., Webb K., Hily J. M., Darbick C. and Scorza R. High transformation efficiency in plum (Prunus domestica L.): a new tool for functional genomics studies in Prunus spp. Mol. Breed. 2008;22:581-591. DOI:10.1007/s11032-008-9200-8

26. Tian L.-N., Canli F. A., Wang X. and Sibbald S. Genetic transformation of Prunus domestica L. Using the hpt gene coding for hygromycin resistance as the selectable marker. Sci. Hortic. 2009;119:339-343. DOI:10.1016/j.scienta.2008.08.024

27. Srinivasan C., Dardick C., Callahan A. and Scorza R. Plum (Prunus domestica) trees transformed with poplar FT1 result in altered architecture, dormancy requirement, and continuous flowering. PLoS One. 2012;7:e40715. DOI:10.1371/journal.pone.0040715

28. Faize M., Faize L., Petri C., Barba-Espin G., Diaz-Vivancos P., Koussa T. et al. Cu/Zn superoxide dismutase and ascorbate peroxidase enhance in vitro shoot multiplication in transgenic plum. J. Plant Physiol. 2013;170:625-632. DOI:10.1016/j.jplph.2012.12.016

29. Escalettes V., Dahuron F., Ravelonandro M. and Dosba F. Utilisation de la transgénose pour l’obtention de pruniers et d’abricotiers exprimant le gène de la protéine capside du plum pox potyvirus. Bull OEPP/EPPO. 1994;24:705-711. DOI:10.1111/j.1365-2338.1994.tb01086.x

30. Yancheva S. D., Druart P. and Watillon B. Agrobacterium mediated transformation of plum (Prunus domestica L.). Acta Hortic. 2002;577:215-217. DOI:10.1371/journal.pone.0050627

31. Petri C. and Scorza R. Factors affecting adventitious regeneration from in vitro leaf explants of improved french plum, the most important dried plum cultivar in the USA. Ann. Appl. Biol. 2010;156:79-89. DOI:10.1111/j.1744-7348.2009.00364.x

32. Mikhailov R. V. and Dolgov S. V. Transgenic plum (Prunus domestica L.) plants obtained by Agrobacterium-mediated transformation of leaf explants with various selective agents. Acta Hortic. 2007;738:613-623. DOI:10.17660/ActaHortic.2007.738.80

33. Sidorova T., Mikhailov R., Pushin A., Miroshnichenko D. and Dolgov S. V. A non-antibiotic selection strategy uses the phosphomannose-isomerase (PMI) gene and green fluorescent protein (GFP) gene for Agrobacterium-mediated transformation of Prunus domestica L. leaf explants. Plant Cell Tiss. Organ. Cult. 2017;128:197-209. DOI:10.1007/s11240-016-1100-8

34. Dolgov S., Mikhaylov R., Serova T., Shulga O. and Firsov A. Pathogen- derived methods for improving resistance of transgenic plums (Prunus domestica L.) for Plum pox virus infection. Julius Kühn Arch. 2010;427:133-140.

35. Jacoboni, A., and Standardi, A. La moltiplicazione “in vitro” del melo cv. Wellspur. Rivista della Ortoflorofrutticoltura Italiana. 1982;66:217-229.

36. Murashige T. and Skoog F. A revised medium for rapid growth bio assays with tobacco tissue culture. Physiol. Plantarum. 1962;15:473-497. DOI:10.1111/j.1399-3054.1962.tb08052.x

37. Lazo G. R., Stein P. A. and Ludwig R. A. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nat. Biotechnol. 1991;9:963-967. DOI:10.1038/nbt1091-967

38. Jeff erson R. A., Kavanagh T. A. and Bevan M. W. GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987;6:3901-3907. DOI:10.1002/j.1460-2075.1987.tb02730.x

39. Rogers S. and Bendich A. “Extraction of total cellular DNA from plants, algae and fungi,” in Plant Molecular Biology Manual, eds S. Gelvin and R. Schiperoort (Dordrecht: Springer), 1994, 183-190.

40. Meisel, L., Fonseca, B., Gonzalez, S., Baeza-Yates, R., Cambiazo, V., Campos, R., et al. A rapid and efficient method for high quality total RNA from peaches (Prunus persica) for functional genomics analyses. Biol. Res. 2005;38:83-88. DOI:10.4067/S0716-97602005000100010

41. Cirilli M., Geuna F., Babini A. R., Bozhkova V., Catalano L., Cavagna B. et al. Fighting sharka in peach: current limitations and future perspectives. Front. Plant Sci. 2016;7:1290. DOI:10.3389/fpls.2016.01290

42. Prieto H. “Genetic transformation strategies in fruit crops,” in Genetic Transformation, ed. M. Alvarez (Chile: National Institute of Agriculture), 2011, 81-100. DOI:10.5772/20887

43. Song, G. Q., Sink, K. C., Walworth, A. E., Cook, M. A., Allison, R. F., and Lang, G. A. Engineering cherry rootstocks with resistance to Prunus necrotic ring spot virus through RNAi-mediated silencing. Plant Biotechnol. J. 2013;11:702-708. DOI:10.1111/pbi.12060

44. Ravelonandro M., Scorza R., Hily J. M., and Briard P. Th e effi ciency of RNA interference for conferring stable resistance to plum pox virus. Plant Cell Tiss. Organ. Cult. 2014;118:347-356. DOI:10.1007/s11240-014-0487-3

45. Zhao D. and Song G. Q. Rootstock-to-scion transfer of transgene-derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry. Plant Biotechnol. J. 2014;12:1319-1328. DOI:10.1111/pbi.12243

46. Malinowski T., Cambra M., Capote N., Zawadzka B., Gorris M. T., Scorza R. et al. Field trials of plum clones transformed with the Plum pox virus coat protein (PPV-cp) gene. Plant Dis. 2006;90:1012-1018. DOI:10.1094/PD-90-1012

47. Lemgo G. N. Y., Sabbadini S., Pandolfi ni T. and Mezzetti B. Biosafety considerations of RNAi-mediated virus resistance in fruit-tree cultivars and in rootstock. Transgenic Res. 2013;22:1073- 1088. DOI:10.1007/s11248-013-9728-1

48. Haroldsen V. M., Chi-Ham C. L., and Bennett A. B. (). Transgene mobilization and regulatory uncertainty for non-GE fruit products of transgenic rootstocks. J. Biotechnol. 2012;161:349-353. DOI:10.1016/j.jbiotec.2012.06.017

49. Flachowsky H., Trankner C., Szankowski I., Waidmann S., Hanke M., Treutter D. et al. (). RNA-mediated gene silencing signals are not graft transmissible from the rootstock to the scion in greenhouse-grown apple plants Malus sp. Int. J. Mol. Sci. 2012;13:9992-10009. DOI:10.3390/ijms13089992

50. Patil B. L. and Fauquet C. M. Light intensity and temperature affect systemic spread of silencing signal in transient agroinfiltration studies. Mol. Plant Pathol. 2015;16:484-494. DOI:10.1111/mpp.12205

51. Lansac M., Eyquard J. P., Salvador B., García-Almodóvara J. A., Le Gall O., Decroocq V. et al. Application of GFP-tagged Plum pox virus to study Prunus-PPV interactions at the whole plant and cellular levels. J. Virol. Methods. 2005;129:125- 133. DOI:10.1016/j.jviromet.2005.05.016

52. Sochor J., Krška B., Polák J. and Juriková T. Th einfluence of virus infections on antioxidant levels in the GM plum variety Honey Sweet (Prunus domestica L.). Potravinárstvo 2015;9:195-200

53. Krška B., Gogolková K., Horsáková J. and Polák J. Effects of economically important virus diseases on the expression of some pomological traits and nutritional compounds in GM plum cultivar HoneySweet (Prunus domesica L.). Hort. Sci. 2017;44:1-5. DOI:10.17221/30/2015-HORTSCI

54. Petri C., Alburquerque N., Faize M., Scorza R. and Dardick, C. Current achievements and future directions in genetic engineering of European plum (Prunus domestica L.). Transgenic Res. 2018;27:225-240. DOI:10.1007/s11248-018-0072-3

55. Malnoy M., Ewa E., Boresjza-Wysocka E. E., Norelli J. L., Flaishman M. A., Gidoni D. et al. Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene. Tree Genet. Genomes. 2010;6:423-433. DOI:10.1007/s11295-009-0260-7

56. Matsuda N., Gao M., Isuzugawa K., Takashina T. and Nishimura K. Development of an Agrobacterium-mediated transformation method for pear (Pyrus communis L.) with leafsection and axillary shoot-meristem explants. Plant Cell Rep. 2005;24:45-51. DOI:10.1007/s00299-005-0924-1

57. Yancheva S., Yablowicz Z., Golubowicz S. and Perl, A. Effi cient Agrobacterium-mediated transformation and recovery of transgenic fi g (Ficus carica L.) plants. Plant Sci. 2005;168:1433-1441. DOI:10.1016/j.plantsci.2004.12.007

58. Cervera M., Navarro A., Navarro L., and Pen´a L. Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration. Tree Physiol. 2008;28:55-66. DOI:10.1093/treephys/28.1.55

59. Orbovic´ V., Shankar A., Peeples M., Hubbard C., and Zale J. “Citrus transformation using mature citrus explants,” in Agrobacterium Protocols, ed.K. Wang (New York, NY: Springer Science and Business Media), 2015, 259-273.

60. Zanek M. C., Reyes C. A., Cervera M., Pen˜a E. J., Vela´zquez K., Costa N. et al. Genetic transformation of sweet orange with the coat protein gene of Citrus psorosis virus and evaluation of resistance against the virus. Plant Cell Rep. 2008;27:57-66. DOI:10.1007/s00299-007-0422-8

61. Scorza R., Ravelonandro M., Callahan A., Zagrai I., Polak J., Malinowski T. et al. “Honey Sweet” (C5), the first genetically engineered plum pox virus — resistant plum (Prunus domestica L.) cultivar. HortScience. 2016;51:601-603. DOI:10.21273/HORTSCI.51.5.601

62. Petri C., Alburquerque N. and Burgos L. (2015). “Apricot (Prunus armeniaca L.),” in Agrobacterium Protocols, ed. K. Wang (New York: Springer), 111-119. DOI:10.1007/978-1-4939-1658-0_10

63. Ramesh S. A., Kaiser B. N., Franks T., Collins G. and Sedgley M. Improved method sin Agrobacterium–mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols. Plant Cell Rep. 2006;25:821-828. DOI:10.1007/s00299-006-0139-0

64. Song G. Q. “Cherry,” in Agrobacterium Protocols, ed. K. Wang (New York, NY: Springer), 2015:133-142. DOI:10.1007/978-1-4939-1658-0_12

65. Sabbadini S., Pandolfi ni T., Girolomini L., Molesini B. and Navacchi O. “Peach (Prunus persica L.),” in Agrobacterium Protocols, ed. K. Wang (New York, NY: Springer), 2015, 205-215. DOI:10.1007/978-1-4939-1658-0_17

66. Sidorova T., Pushin A., Miroshnichenko D. and Dolgov S. V. Generation of transgenic rootstock plum ((Prunus pumila L.×P. salicina Lindl.) x (P. cerasifera Ehrh.)) using hairpin-RNA construct for resistance to the Plum pox virus. Agronomy. 2018;8:2. DOI:10.3390/agronomy8010002