A rare transgenic event of rice with Agrobacterium binary vector backbone integration at the right T-DNA border junction

Abstract

Agrobacterium tumefaciens precisely transfers the transferred DNA (T-DNA) between the right border (RB) and the left border (LB) and integrates it in the plant genome. However, transfer of the binary vector backbone (BVB) at the LB junction has been frequently reported. Here, we describe a complex T-DNA integration in the rice transgenic event CG27 with BVB at the RB junction. Amplification of an RB-plant DNA junction fragment by genome walking and its sequencing revealed that the T-DNA portion ended at the 1st bp of RB, but is followed by a binary vector backbone (BVB) along with an RB-flanking T-DNA portion, both in an inverse orientation. This is followed by a 5-bp filler sequence and the rice chromosome 1 sequence. The configuration of the BVB integration at the RB junction was independently confirmed by PCR. Disruption of native chromosome 1 sequence was confirmed by PCR with chromosome 1-specific primers designed from either side of the T-DNA integration site. BVB sequence close to the RB is transferred and integrated into the CG27 plant genome by a mechanism that does not involve LB-skipping and RB read-through. Therefore, a check for BVB sequences at both LB and RB junctions in a transgenic crop is important in biosafety evaluation.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Abbreviations

T-DNA:

Transferred DNA

RB:

Right border

LB:

Left border

BVB:

Binary vector backbone

MS:

Murashige and Skoog medium

References

  1. Albright LM, Yanofsky MF, Leroux B, Ma DQ, Nester EW (1987) Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA. J Bacteriol 169:1046–1055

    CAS  Article  Google Scholar 

  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    CAS  Article  Google Scholar 

  3. Brunk CF, Jones KC, James TW (1979) Assay for nanogram quantities of DNA in cellular homogenates. Anal Biochem 92:497–500

    CAS  Article  Google Scholar 

  4. De Buck S, De Wilde C, Van Montagu M, Depicker A (2000) T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation. Mol Breed 6:459–468

    Article  Google Scholar 

  5. De Vos G, Zambryski P (1989) Expression of Agrobacterium nopaline-specific VirD1, VirD2, and VirC1 proteins and their requirement. Mol Plant Microbe Interact 2:43–52

    Article  Google Scholar 

  6. Gang H, Liu G, Zhang M, Zhoa Y, Jiang J, Chen S (2019) Comprehensive characterization of T-DNA integration induced chromosomal rearrangement in a birch T-DNA mutant. BMC Genomics. https://doi.org/10.1186/s12864-019-5636-y

    Article  PubMed  PubMed Central  Google Scholar 

  7. Gelvin SB (2017) Integration of Agrobacterium T-DNA into the plant genome. Annu Rev Genet 51:195–217

    CAS  Article  Google Scholar 

  8. Gheysen G, Van Montagu M, Zambryski P (1987) Integration of Agrobacterium tumefaciens transfer DNA (T-DNA) involves rearrangements of target plant DNA sequences. Proc Natl Acad Sci USA 17:6169–6173

    Article  Google Scholar 

  9. Gheysen G, Villarroel R, Van Montagu M (1991) Illegitimate recombination in plants: a model for T-DNA integration. Genes Dev 5:287–297

    CAS  Article  Google Scholar 

  10. Guo M, Ye J, Gao D, Xu N, Yang J (2019) Agrobacterium-mediated horizontal gene transfer: Mechanism, biotechnological application, potential risk and forestalling strategy. Biotechnol Adv 37:259–270

    CAS  Article  Google Scholar 

  11. Kannan P, Parameswari C, Prasanyaselvam K, Sridevi G, Veluthambi K (2017) Introgression of sheath blight disease tolerance from the transgenic rice event Pusa Basmati1-CG27 to the variety White Ponni through backcross breeding. Indian J Genet 77:501–507

    CAS  Google Scholar 

  12. Kim SR, Lee J, Jun SH, Park S, Kang HG, Kwon S, An G (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52:761–773

    CAS  Article  Google Scholar 

  13. Kleinboelting N, Huep G, Appelhagen I, Viehoever P, Li Y, Weisshaar B (2015) The structural features of thousands of T-DNA insertion sites are consistent with a double-strand break repair-based insertion mechanism. Mol Plant 8:1651–1664

    CAS  Article  Google Scholar 

  14. Kononov ME, Bassuner B, Gelvin SB (1997) Integration of T-DNA binary vector ‘backbone’sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J 11:945–957

    CAS  Article  Google Scholar 

  15. Laufs P, Autran D, Traas J (1999) A chromosomal paracentric inversion associated with T-DNA integration in Arabidopsis. Plant J 18:131–139

    CAS  Article  Google Scholar 

  16. Majhi BB, Shah JM, Veluthambi K (2014) A novel T-DNA integration in rice involving two interchromosomal translocations. Plant Cell Rep 33:929–944

    CAS  Article  Google Scholar 

  17. Martineau B, Voelker TA, Sanders RA (1994) On defining T-DNA. Plant Cell 6:1032

    Article  Google Scholar 

  18. Mayerhofer R, Koncz-Kalman Z, Nawrath C, Bakkeren G, Crameri A, Angelis K, Redei GP, Schell J, Hohn B, Koncz C (1991) T-DNA integration: a mode of illegitimate recombination in plants. EMBO J 10:697–704

    CAS  Article  Google Scholar 

  19. Nacry P, Camilleri C, Courtial B, Caboche M, Bouchez D (1998) Major chromosomal rearrangements induced by T-DNA transformation in Arabidopsis. Genetics 149:641–650

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Nicolia A, Ferradini N, Veronesi F, Rosellini D (2017) An insight into T-DNA integration events in Medicago sativa. Int J Mol Sci. https://doi.org/10.3390/ijms18091951

    Article  PubMed  PubMed Central  Google Scholar 

  21. Ohba T, Yoshioka Y, Machida C, Machida Y (1995) DNA rearrangement associated with the integration of T-DNA in tobacco: an example for multiple duplications of DNA around the integration target. Plant J 7:157–164

    CAS  Article  Google Scholar 

  22. Ramanathan V, Veluthambi K (1995) Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of T L-DNA. Plant Mol Biol 28:1149–1154

    CAS  Article  Google Scholar 

  23. Ramanathan V, Veluthambi K (1996) Analysis of octopine left border-directed DNA transfer from Agrobacterium to plants. J Biosci 21:45–56

    CAS  Article  Google Scholar 

  24. Rogers SO, Bendich AJ (1988) Extraction of DNA from plant tissues. In: Gelvin SB, Schilperoort RA, Verma DPS (eds) Plant molecular biology manual, vol A6. Kluwer, Dordrecht, pp 1–10

    Google Scholar 

  25. Sha Y, Li S, Pei Z, Luo L, Tian Y, He C (2004) Generation and flanking sequence analysis of a rice T-DNA tagged population. Theor Appl Genet 108:306–314

    CAS  Article  Google Scholar 

  26. Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23:1087–1088

    CAS  Article  Google Scholar 

  27. Sridevi G, Parameswari C, Sabapathi N, Raghupathy V, Veluthambi K (2008) Combined expression of chitinase and β-1, 3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani. Plant Sci 175:283–290

    CAS  Article  Google Scholar 

  28. Stachel SE, Timmerman B, Zambryski P (1987) Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5′ virD gene products. EMBO J 6:857–863

    CAS  Article  Google Scholar 

  29. van der Graaff E, den Dulk-Ras A, Hooykaas PJ (1996) Deviating T-DNA transfer from Agrobacterium tumefaciens to plants. Plant Mol Biol 31:677–681

    Article  Google Scholar 

  30. Veluthambi K, Ream W, Gelvin SB (1988) Virulence genes, borders and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens. J Bacteriol 170:1523–1532

    CAS  Article  Google Scholar 

  31. Wenck A, Czakó M, Kanevski I, Márton L (1997) Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation. Plant Mol Biol 34:913–922

    CAS  Article  Google Scholar 

  32. Winans SC, Ebert PR, Stachel SE, Gordon MP, Nester EW (1986) A gene responsible for Agrobacterium virulence is homologous to a family of positive regulatory loci. Proc Natl Acad Sci USA 83:8278–8282

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India [Project entitled “Development of sheath blight disease-resistant transgenic rice: Resistance tests in PR-protein-expressing transgenic rice and discovery of new RNA silencing strategies”, Project number: F.No: BT/PR6466/COE/34/16/2012]. Indian National Science Academy, New Delhi is acknowledged for the Senior Scientist Fellowship to K.V. Dr. P. Sundaresan, Aravind Medical Research Foundation, Madurai is thanked for DNA Sequencing Service.

Author information

Affiliations

Authors

Contributions

KV and VR conceived and designed all experiments, analyzed the data and wrote the manuscript. GS generated the rice transgenic event CG27. PK did a part of genome walking experiments and nucleotide sequence analysis. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Karuppannan Veluthambi.

Ethics declarations

Conflict of interest

The authors declare that there are no potential conflicts of interest regarding the publication of this paper. All authors have read and approved this version of the article and due care has been taken to ensure the correctness of the data reported in the paper.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rajapriya, V., Kannan, P., Sridevi, G. et al. A rare transgenic event of rice with Agrobacterium binary vector backbone integration at the right T-DNA border junction. J. Plant Biochem. Biotechnol. (2021). https://doi.org/10.1007/s13562-021-00647-3

Download citation

Keywords

  • Biosafety
  • Complex T-DNA integration
  • Event-specific PCR
  • Genome walking
  • Native site disruption
  • Right border
  • Vector backbone integration