Skip to main content
SpringerLink
Log in
Book cover

Antiviral Resistance in Plants pp 11–25Cite as

Insertion of Epitope Tag into NB-LRR Class Plant Virus Resistance Protein

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2028))

Abstract

It is prerequisite to detect plant disease resistance proteins for studying the function of the proteins. Numerous studies have used epitope tags fused to either N- or C-terminus for the detection of resistance proteins. However, some resistance proteins do not tolerate the terminal fusions of epitope tags. In this chapter, we provide a protocol for searching the protein regions in which the inserted epitope tag does not affect the protein function. In the protocol, we first perform an in silico search to select the insertion site candidates and then insert there a short sequence containing restriction sites to find out the sites, in which the insertion does not affect the protein function. Epitope tags are inserted into the experimentally selected sites to produce a functional protein with an epitope tag.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Whitham S, Dinesh-Kumar SP, Choi D, Hehl R, Corr C, Baker B (1994) The product of the tobacco mosaic virus resistance gene N: similarity to toll and the interleukin-1 receptor. Cell 78(6):1101–1115. https://doi.org/10.1016/0092-8674(94)90283-6

    Article  CAS  PubMed  Google Scholar 

  2. Les EF, Holzberg S, Calderon-Urrea A, Handley V, Axtell M, Corr C, Baker B (1999) The helicase domain of the TMV replicase proteins induces the N-mediated defence response in tobacco. Plant J 18(1):67–75. https://doi.org/10.1046/j.1365-313X.1999.00426.x

    Article  Google Scholar 

  3. Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP (2002) Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30(4):415–429. https://doi.org/10.1046/j.1365-313X.2002.01297.x

    Article  CAS  PubMed  Google Scholar 

  4. Liu Y, Schiff M, Dinesh-Kumar SP (2004) Involvement of MEK1 MAPKK, NTF6 MAPK, WRKY/MYB transcription factors, COI1 and CTR1 in N-mediated resistance to tobacco mosaic virus. Plant J 38(5):800–809. https://doi.org/10.1111/j.1365-313X.2004.02085.x

    Article  CAS  PubMed  Google Scholar 

  5. Liu Y, Schiff M, Serino G, Deng X-W, Dinesh-Kumar SP, Martin GB, Chai J (2002) Role of SCF ubiquitin-ligase and the COP9 signalosome in the N gene-mediated resistance response to tobacco mosaic virus. Plant Cell 14(7):1483–1496. https://doi.org/10.1105/tpc.002493.pathogen

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jin H, Liu Y, Yang KY, Kim CY, Baker B, Zhang S (2003) Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco. Plant J 33(4):719–731. https://doi.org/10.1046/j.1365-313X.2003.01664.x

    Article  CAS  PubMed  Google Scholar 

  7. Hoser R, Zurczak M, Lichocka M et al (2013) Nucleocytoplasmic partitioning of tobacco N receptor is modulated by SGT1. New Phytol 200(1):158–171. https://doi.org/10.1111/nph.12347

    Article  CAS  PubMed  Google Scholar 

  8. Caplan JL, Mamillapalli P, Burch-Smith TM, Czymmek K, Dinesh-Kumar SP (2008) Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector. Cell 132:449–462

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Dinesh-Kumar SP, Baker BJ (2000) Alter-natively spliced N resistance gene transcripts: their possible role in tobacco mosaic virus resistance. Proc Natl Acad Sci 97(4):1908–1913. https://doi.org/10.1073/pnas.020367497

    Article  CAS  Google Scholar 

  10. Mestre P, Baulcombe DC (2006) Elicitor-mediated oligomerization of the tobacco N disease resistance protein. Plant Cell 18:491–501

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rairdan GJ, Moffett P (2006) Distinct domains in the ARC region of the potato resistance protein Rx mediate LRR binding and inhibition of activation. Plant Cell 18:2082–2093

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Rairdan GJ, Collier SM, Sacco MA, Baldwin TT, Boettrich T, Moffett P (2008) The coiled-coil and nucleotide binding domains of the potato Rx disease resistance protein function in pathogen recognition and signaling. Plant Cell 20:739–751

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Moffett P, Farnham G, Peart J, Baulcombe DC (2002) Interaction between domains of a plant NBS-LRR protein in disease resistance-related cell death. EMBO J 21:4511–4519

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tameling WI, Baulcombe DC (2007) Physical association of the NB-LRR resistance protein Rx with a Ran GTPase-activating protein is required for extreme resistance to Potato virus X. Plant Cell 19:1682–1694

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sacco MA, Mansoor S, Moffett P (2007) A RanGAP protein physically interacts with the NB-LRR protein Rx, and is required for Rx-mediated viral resistance. Plant J 52:82–93

    Article  CAS  PubMed  Google Scholar 

  16. Takahashi H, Shoji H, Ando S, Kanayama Y, Kusano T, Takeshita M, Suzuki M, Masuta C (2012) RCY1 -mediated resistance to Cucumber mosaic virus is regulated by LRR domain-mediated interaction with CMV(Y) following degradation of RCY1. Mol Plant-Microbe Interact 25:1171–1185

    Article  CAS  PubMed  Google Scholar 

  17. Jarvik JW, Telmer CA (1998) Epitope tagging. Annu Rev Genet 32:601–618

    Article  CAS  PubMed  Google Scholar 

  18. Sekine K-T, Tomita R, Takeuchi S et al (2012) Functional differentiation in the leucine-rich repeat domains of closely related plant virus-resistance proteins that recognize common avr proteins. Mol Plant-Microbe Interact 25:1219–1229

    Article  CAS  PubMed  Google Scholar 

  19. Nelson BD, Manoil C, Traxler B (1997) Insertion mutagenesis of the lac repressor and its implications for structure-function analysis. J Bacteriol 179(11):3721–3728

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tomita R, Sekine K-T, Mizumoto H, Sakamoto M, Murai J, Kiba A, Hikichi Y, Suzuki K, Kobayashi K (2011) Genetic basis for the hierarchical interaction between Tobamovirus spp. and L resistance gene alleles from different pepper species. Mol Plant-Microbe Interact 24:108–117

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported in part by JSPS KAKENHI Grant Numbers 21380032, 24658044, 26292026 and 15K14664.

Author information

Authors and Affiliations

  1. Faculty of Agriculture, Ehime University, Matsuyama, Ehime, Japan

    Kappei Kobayashi

  2. Faculty of Agriculture, University of the Ryukyus, Nishihara, Okinawa, Japan

    Reiko Tomita & Ken-Taro Sekine

Authors
  1. Kappei Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Reiko Tomita
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ken-Taro Sekine
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kappei Kobayashi .

Editor information

Editors and Affiliations

  1. Faculty of Agriculture, Ehime University, Matsuyama, Ehime, Japan

    Kappei Kobayashi

  2. Faculty of Agriculture, Ehime University, Matsuyama, Ehime, Japan

    Masamichi Nishiguchi

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kobayashi, K., Tomita, R., Sekine, KT. (2019). Insertion of Epitope Tag into NB-LRR Class Plant Virus Resistance Protein. In: Kobayashi, K., Nishiguchi, M. (eds) Antiviral Resistance in Plants. Methods in Molecular Biology, vol 2028. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9635-3_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9635-3_2

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9634-6

  • Online ISBN: 978-1-4939-9635-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation