Experimental and Applied Acarology

, Volume 67, Issue 2, pp 269–287 | Cite as

Transgene expression in tick cells using Agrobacterium tumefaciens

  • Erik Machado-Ferreira
  • Emilia Balsemão-Pires
  • Gabrielle Dietrich
  • Andrias Hojgaard
  • Vinicius F. Vizzoni
  • Glen Scoles
  • Lesley Bell-Sakyi
  • Joseph Piesman
  • Nordin S. Zeidner
  • Carlos A. G. SoaresEmail author


Ticks transmit infectious agents to humans and other animals. Genetic manipulation of vectors like ticks could enhance the development of alternative disease control strategies. Transgene expression using the phytopathogen Agrobacterium tumefaciens has been shown to promote the genetic modification of non-plant cells. In the present work we developed T-DNA constructs for A. tumefaciens to mediate transgene expression in HeLa cells as well as Rhipicephalus microplus tick cells. Translational fusions eGfp:eGfp or Salp15:eGfp, including the enhanced-green fluorescent protein and the Ixodes scapularis salivary factor SALP15 genes, were constructed using the CaMV 35S (cauliflower mosaic virus) promoter, “PBm” tick promoter (R. microplus pyrethroid metabolizing esterase gene) or the Simian Virus SV40 promoter. Confocal microscopy, RT-PCR and Western-blot assays demonstrated transgene(s) expression in both cell lines. Transgene expression was also achieved in vivo, in both R. microplus and I. scapularis larvae utilizing a soaking method including the A. tumefaciens donor cells and confirmed by nested-RT-PCR showing eGfp or Salp15 poly-A-mRNA(s). This strategy opens up a new avenue to express exogenous genes in ticks and represents a potential breakthrough for the study of tick-host pathophysiology.


Transgene expression Tick cells Agrobacterium tumefaciens Rhipicephalus microplus Ixodes scapularis 



Erik Machado-Ferreira’s fellowship was supported by CDC and the Brazilian agency CAPES in the Departamento de Genética-Instituto de Biologia (Universidade Federal do Rio de Janeiro). E. Balsemão-Pires was supported by a PhD fellowship from CAPES/Brazil and a SWE fellowship from CNPq/Brazil. The BME/CTVM2 cell line was provided by the Tick Cell Biobank. The anti-Salp15 antibody was supplied by Dr. Amy Ullmann-Moore of the CDC.

Supplementary material

10493_2015_9949_MOESM1_ESM.pdf (309 kb)
Supplemental fig. 1 Confocal microscopy of N. benthamiana leaves infiltrated with A. tumefaciens pP35S-eGFPeGFP. Free eGFP (green) and chlorophyll-a (red) emissions captured at 505-530 nm and 685nm, respectively, are presented in the left-side and central panels. Merged images are displayed in the right-side panels. Emission for the infiltrations with T-DNA construct with P35S:eGfp:eGfp (eGFP T-DNA) and with bacteria-free buffer (Mock transformation) are shown in each figure row. Bar = 50 µM (PDF 309 kb)


  1. Anguita J, Ramamoorthi N, Hovius JWR, Das S, Thomas V, Persinski R, Conze D, Askenase PW, Rincón M, Kantor FS, Fikrig E (2002) Salp15, an Ixodes scapularis salivary protein, inhibits CD4+ T cell activation. Immunity 16:849–859CrossRefPubMedGoogle Scholar
  2. Baldridge GD, Kurtti TJ, Burkhardt N, Baldridge AS, Nelson CM, Oliva AS, Munderloh UG (2007) Infection of Ixodes scapularis ticks with Rickettsia monacensis expressing green fluorescent protein: a model system. J Invertebr Pathol 94:163–174PubMedCentralCrossRefPubMedGoogle Scholar
  3. Bell-Sakyi L (2004) Ehrlichia ruminantium grows in cell lines from four ixodid tick genera. J Comp Pathol 130:285–293CrossRefPubMedGoogle Scholar
  4. Binns AN, Thomashow MF (1988) Cell biology of Agrobacterium infection and transformation of plants. Annu Rev Microbiol 42:575–606CrossRefGoogle Scholar
  5. Black WC, Piesman J (1994) Phylogeny of hard- and soft-tick taxa (Acari: Ixodidae) based on mitochondrial 16S rDNA sequences. Proc Natl Acad Sci USA 91:10034–10038PubMedCentralCrossRefPubMedGoogle Scholar
  6. Brossard M, Wikel SK (2004) Tick immunobiology. Parasitology 124:161–176CrossRefGoogle Scholar
  7. Bundock P, Hooyokaas PJ (1996) Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc Natl Acad Sci USA 93:15272–15275PubMedCentralCrossRefPubMedGoogle Scholar
  8. De Buck S, Jacobs A, Van Montagu M, Depicker A (1998) Agrobacterium tumefaciens transformation and cotransformation of Arabidopsis thaliana root explants and tobacco protoplasts. Mol Plant Microbe Interact 11:449–457CrossRefPubMedGoogle Scholar
  9. de Groot MJ, Bundock P, Hooyokaas PJ, Beijersbergen AG (1998) Agrobacterium tumefaciens mediated transformation of filamentous fungi. Nat Biotechnol 16:839–842CrossRefPubMedGoogle Scholar
  10. de la Fuente J, Blouin EF, Manzano-Roman R, Naranjo V, Almazán C et al (2007a) Functional genomic studies of tick cells in response to infection with the cattle pathogen, Anaplasma marginale. Genomics 90:712–722CrossRefPubMedGoogle Scholar
  11. de la Fuente J, Kocan KM, Almazan C, Blouin EF (2007b) RNA interference for the study and genetic manipulation of ticks. Trends Parasitol 23:427–433CrossRefPubMedGoogle Scholar
  12. de Silva AM, Tyson KR, Pal U (2009) Molecular characterization of the tick-Borrelia interface. Front Biosci 14:3051–3063CrossRefGoogle Scholar
  13. Fladung M (1999) Gene stability in transgenic aspen (Populus)—I: flanking DNA sequences and T-DNA structure. Mol Gen Genet 260:574–581CrossRefPubMedGoogle Scholar
  14. Francischetti IMB, Sa-Nunes A, Mans BJ, Santos IM, Ribeiro JMC (2009) The role of saliva in tick feeding. Front Biosci 14:2051–2088CrossRefGoogle Scholar
  15. Gaudin V, Vrain T, Jouanin L (1994) Bacterial genes modifying hormonal balances in plants. Plant Physiol Biochem 32:11–29Google Scholar
  16. Gillespie JJ, Brayton KA, Williams KP, Diaz MA, Brown WC, Azad AF, Sobral BW (2010) Phylogenomics reveals a diverse Rickettsiales type IV secretion system. Infect Immun 78:1809–1823PubMedCentralCrossRefPubMedGoogle Scholar
  17. Guerrero FD, Nene VM (2008) Gene structure and expression of a pyrethroid-metabolizing esterase, CzEst9, from a pyrethroid resistant Mexican population of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). J Med Entomol 45:677–685PubMedGoogle Scholar
  18. Hackstadt T (1996) The biology of rickettisae. Infect Agents Dis 5:127–143PubMedGoogle Scholar
  19. Haas JH, Moore LW, Ream W, Manulis S (1995) Universal primers for detection of pathogenic Agrobacterium strains. Appl Environ Microbiol 6:2879–2884Google Scholar
  20. Herrera-Estrella L, Simpson J, Martinez-Trujillo M (2005) Transgenic plants: an historical perspective. Methods Mol Biol 286:3–32PubMedGoogle Scholar
  21. Hojgaard A, Biketov SF, Shtannikov AV, Zeidner NS, Piesman J (2009) Molecular identification of Salp15, a key salivary gland protein in the transmission of lyme disease spirochetes, from Ixodes persulcatus and Ixodes pacificus (Acari: Ixodidae). J Med Entomol 46:1458–1463Google Scholar
  22. Karime M, Inze D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195CrossRefGoogle Scholar
  23. Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396CrossRefGoogle Scholar
  24. Kunik T, Tzifira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky C (2001) Genetic transformation of HeLa cells by Agrobacterium. Proc Natl Acad Sci USA 98:1871–1876PubMedCentralCrossRefPubMedGoogle Scholar
  25. Kurtenbach K, Sewell HS, Ogden NH, Randolph SE, Nuttall PA (1998) Serum complement sensitivity as a key factor in Lyme disease ecology. Infect Immun 66:1248–1251PubMedCentralPubMedGoogle Scholar
  26. Kurtti TJ, Mattila JT, Herron MJ, Felsheim RF, Baldridge GD, Burkhardt NY, Blazar BR, Hackett PB, Meyer JM, Munderloh UG (2008) Transgene expression and silencing in tick cell line: a model system for functional tick genomics. Insect Biochem Mol Biol 38:963–968PubMedCentralCrossRefPubMedGoogle Scholar
  27. Lacroix B, Tzfira T, Vainstein A, Citovsky V (2006) A case of promiscuity: Agrobacterium´s endless hunt for new partners. Trends Genet 22:29–37CrossRefPubMedGoogle Scholar
  28. Lo N, Beninati T, Sassera D, Bouman EA, Santagati S, Gern L, Sambri V, Masuzawa T, Gray JS, Jaenson TG, Bouattour A, Kenny MJ, Guner ES, Kharitonenkov IG, Bitam I, Bandi C (2006) Widespread distribution and high prevalence of an alpha-proteobacterial symbiont in the tick Ixodes ricinus. Environ Microbiol 7:1280–1287CrossRefGoogle Scholar
  29. Loyter A, Rosenbluh J, Zakai N, Li J, Kozlovsky SV, Tzfira T, Citovsky V (2005) The plant VirE2 interacting protein 1: a molecular link between the Agrobacterium T-complex and the host cell chromatin? Plant Physiol 138:1318–1321PubMedCentralCrossRefPubMedGoogle Scholar
  30. Mattila JT, Burkhardt NY, Hutcheson HJ, Munderloh UG, Kurtti TJ (2007) Isolation of cell lines and a rickettsial endosymbiont from the soft tick Carios capensis (Acari: Argasidae: Ornithodorinae). J Med Entomol 44:1091–1101CrossRefPubMedGoogle Scholar
  31. McCullen CA, Binns AN (2006) Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annu Rev Cell Dev Biol 22:101–127CrossRefPubMedGoogle Scholar
  32. Mougel C, Cournoyer B, Nesme X (2001) Novel tellurite-amended media and specific chromosomal and TI plasmid probes for direct analysis of soil popuations of Agrobacterium biovars 1 and 2. Appl Environ Microbiol 67:65–74Google Scholar
  33. Pal U, Li X, Wang T, Montgomery RR, Ramamoorthi N, Desilva AM, Bao F, Yang X, Pypaert M, Pradhan D, Kantor FS, Telford S, Anderson JF, Fikrig E (2004) TROSPA an Ixodes scapularis receptor for Borrelia burgdorferi. Cell 199:457–468CrossRefGoogle Scholar
  34. Pelczar P, Kalck V, Gomez D, Hohn B (2004) Agrobacterium proteins VirD2 e VirE2 mediate precise integration of synthetic T-DNA complex in mammalian cells. EMBO Rep 5:632–637PubMedCentralCrossRefPubMedGoogle Scholar
  35. Piesman J (1993) Standard system for infecting ticks (Acari: Ixodidae) with the Lyme disease spirochete, Borrelia burgdorferi. J Med Entomol 30:199–203CrossRefPubMedGoogle Scholar
  36. Policastro PF, Schwan TG (2003) Experimental infection of Ixodes scapularis larvae (Acari: Ixodidae) by immersion in low passage cultures of Borrelia burgdorferi. J Med Entomol 40:364–370CrossRefPubMedGoogle Scholar
  37. Rai M, Datta K, Parkhi V, Tan J, Oliva N, Chawla HS, Datta SK (2007) Variable T-DNA linkage configuration affects inheritance of carotenogenic transgenes and carotenoid accumulation in transgenic indica rice. Plant Cell Rep 26:1221–1231CrossRefPubMedGoogle Scholar
  38. Ramamoorthi N, Narasimhan S, Pal U, Bao F, Yang XF, Fish D, Anguita J, Norgard MV, Kantor FS, Anderson JF, Koski RA, Fikrig E (2005) The Lyme disease agent exploits a tick protein to infect the mammalian host. Nature 436:573–577PubMedCentralCrossRefPubMedGoogle Scholar
  39. Ribeiro JMC, Francischetti IMB (2003) Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annu Rev Entomol 48:73–88CrossRefPubMedGoogle Scholar
  40. Scherer WF, Syverton JT, Gey GO (1953) HeLa: studies on the propagation in vitro of poliomyelitis viruses—IV: viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix. J Exp Med 97:695–710PubMedCentralCrossRefPubMedGoogle Scholar
  41. Sheng J, Citovisky V (1996) Agrobacterium-plant cell DNA transport: have virulence proteins, will travel. Plant Cell 8:1699–1710PubMedCentralCrossRefPubMedGoogle Scholar
  42. Simser JA, Palmer AT, Fingerle V, Wilske B, Kurtti TJ, Munderloh UG (2002) Rickettsia monacensis sp. nov, a spotted fever group Rickettsia, from ticks (Ixodes ricinus) collected in a European city park. Appl Environ Microbiol 68:4559–4566PubMedCentralCrossRefPubMedGoogle Scholar
  43. Soares CA, Lima CM, Dolan MC, Piesman J, Beard CB, Zeidner NS (2005) Capillary feeding of specific dsRNA induces silencing of the isac gene in nymphal Ixodes scapularis ticks. Insect Mol Biol 14:443–452CrossRefPubMedGoogle Scholar
  44. Sonenshine DE (1993) Biology of ticks, vol 2. Oxford University Press, New York, NYGoogle Scholar
  45. Stachel SE, Messens E, Van Montagu M, Zambryski K (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629CrossRefGoogle Scholar
  46. Tzfira T, Citovsky V (2002) Partners-in-infection: host proteins involved in the transformation of plant cells by Agrobacterium. Trends Cell Biol 12:121–129CrossRefPubMedGoogle Scholar
  47. Tzfira T, Citovsky V (2006) Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr Opin Biotechnol 17:147–154CrossRefPubMedGoogle Scholar
  48. Ullmann AJ, Lima CMR, Guerrero FD, Piesman J, Black WC (2005) Genome size and organization of the blacklegged tick, Ixodes scapularis and the southern cattle tick, Boophilus microplus. Insect Mol Biol 14:217–222CrossRefPubMedGoogle Scholar
  49. Valenzuela JG, Francischetti IMB, Pham VM, Garfield MK, Mather TN, Ribeiro JM (2002) Exploring the sialome of the tick Ixodes scapularis. J Exp Biol 205:2843–2864PubMedGoogle Scholar
  50. Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956CrossRefPubMedGoogle Scholar
  51. Weller SJ, Baldridge GD, Munderloh UG, Noda H, Simser J, Kurtti TJ (1998) Phylogenetic placement of Rickettsiae from the ticks Amblyomma americanum and Ixodes scapularis. J Clin Microbiol 36:1305–1317PubMedCentralPubMedGoogle Scholar
  52. Wikel SK (1996) Host immunity to ticks. Annu Rev Entomol 41:1–22CrossRefPubMedGoogle Scholar
  53. Wikel SK (1999) Tick modulation of host immunity: an important factor in pathogen transmission. Int J Parasitol 29:851–859CrossRefPubMedGoogle Scholar
  54. Winans SC (1992) Two-way chemical signaling in Agrobacterium-plant interactions. Microbiol Rev 56:12–31PubMedCentralPubMedGoogle Scholar
  55. Winston PW, Bates DH (1960) Saturated solutions for the control of humidity in biological research. Ecology 41:232–237CrossRefGoogle Scholar
  56. Zivkovic Z, Esteves E, Almazan C, Daffre S, Nijhof AM (2010) Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus) microplus in response to infection with Anaplasma marginale. BMC Genomics 11:186PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Erik Machado-Ferreira
    • 1
    • 2
  • Emilia Balsemão-Pires
    • 1
  • Gabrielle Dietrich
    • 2
  • Andrias Hojgaard
    • 2
  • Vinicius F. Vizzoni
    • 1
  • Glen Scoles
    • 3
  • Lesley Bell-Sakyi
    • 4
  • Joseph Piesman
    • 2
  • Nordin S. Zeidner
    • 2
  • Carlos A. G. Soares
    • 1
    Email author
  1. 1.Lab. Genética Molecular de Eucariontes e Simbiotes, Departamento de Genética, Instituto de BiologiaUniversidade Federal do Rio de JaneiroRio de JaneiroBrazil
  2. 2.Bacterial Zoonoses Branch, Division of Vector-Borne Infectious DiseasesCenters for Disease Control and PreventionFort CollinsUSA
  3. 3.Agricultural Research ServicesUSDAPullmanUSA
  4. 4.The Pirbright InstitutePirbrightUK

Personalised recommendations