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Replication Assay of Begomovirus in Arabidopsis Protoplasts

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Plant-Virus Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2724))

Abstract

Protoplasts are isolated plant cells from which the cell walls have been removed by treatment with fungal cellulase and macerozyme enzymes, which degrade the primary components of the cell wall. The protoplasts are totipotent, sensitive, and versatile; thereby, they have been extensively used to study signal transduction pathways, cell-autonomous responses, and replication of plant viruses. This system has several advantages over the use of whole plants for viral replication, including a high percentage of infected cells and uncoupling virus movement from replication assays. Here, we describe a simple and efficient protocol for begomovirus transfection and replication in Arabidopsis protoplasts. The method consists of four steps, (i) protoplast isolation, (ii) PEG–calcium transfection of begomovirus infectious clones, (iii) elimination of input plasmid DNA by DpnI digestion, and (iv) quantification of the viral newly synthesized DNA by qPCR. The protoplasts can be transformed efficiently with begomovirus infectious clones, and virus replication can be monitored by the accumulation of nascent viral DNA in the infected protoplasts.

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References

  1. Kanai R, Edwards GE (1973) Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies. Plant Physiol 51:1133–1137. https://doi.org/10.2307/4263286

    Article  CAS  Google Scholar 

  2. Nagata T, Takebe I (1971) Plating of isolated tobacco mesophyll protoplasts on agar medium. Planta 99:12–20. https://doi.org/10.1007/BF00392116

    Article  CAS  Google Scholar 

  3. Nanjareddy K, Arthikala MK, Blanco L, Arellano ES, Lara M (2016) Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis. BMC Biotechnol 16:53. https://doi.org/10.1186/s12896-016-0283-8

    Article  CAS  Google Scholar 

  4. Ren R, Gao J, Yin D et al (2021) Highly efficient leaf base protoplast isolation and transient expression systems for orchids and other important monocot crops. Front Plant Sci 12:626015. https://doi.org/10.3389/fpls.2021.626015

    Article  Google Scholar 

  5. Toriyama HK (1985) Cell suspension and protoplast culture in rice. Plant Sci 41:179–183. https://doi.org/10.1016/0168-9452(85)90086-X

    Article  CAS  Google Scholar 

  6. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572. https://doi.org/10.1038/nprot.2007.199

    Article  CAS  Google Scholar 

  7. Gouveia-Mageste BC, Martins LGC, Dal-Bianco M et al (2021) A plant-specific syntaxin-6 protein contributes to the intracytoplasmic route for the begomovirus CabLCV. Plant Physiol 187:158–173. https://doi.org/10.1093/plphys/kiab252

    Article  CAS  Google Scholar 

  8. Chodon A, Karanthamalai J, Pandi G, Tennyson J (2022) The begomovirus–host interplay in viral replication and movement. In: Gaur RK, Sharma P, Czosnek H (Org) Geminivirus: detection, diagnosis and management, 1st edn. Academic Press, London, p 397–419. https://doi.org/10.1016/B978-0-323-90587-9.00001-8

  9. Kumar RV (2019) Plant antiviral immunity against geminiviruses and viral counter-defense for survival. Front Microbiol 10:1460. https://doi.org/10.3389/fmicb.2019.01460

    Article  Google Scholar 

  10. Wu M, Wei H, Tan H, Pan S, Liu Q, Bejarano ER, Lozano-Durán R (2021) Plant DNA polymerases α and δ mediate replication of geminiviruses. Nat Commun 2021(12):2780. https://doi.org/10.1038/s41467-021-23013-2

    Article  CAS  Google Scholar 

  11. Martins LGC, Raimundo GAS, Ribeiro NGA et al (2020) A Begomovirus nuclear shuttle protein-interacting immune hub: hijacking host transport activities and suppressing incompatible functions. Front Plant Sci 11:398. https://doi.org/10.3389/fpls.2020.00398

    Article  Google Scholar 

  12. Silva FA, Raimundo GAS., Fontes EBP (2022) Begomovirus:host protein-protein interactions in intracellular virus movement. In: Gaur RK, Sharma P, Czosnek H (Org) Geminivirus: detection, diagnosis and management. 1st edn. Academic Press, London, p. 347–356. https://doi.org/10.1016/B978-0-323-90587-9.00023-7

  13. Wang A (2021) Cell-to-cell movement of plant viruses via plasmodesmata: a current perspective on potyviruses. Curr Opin Virol 48:10–16. https://doi.org/10.1007/s10265-014-0683-6

    Article  CAS  Google Scholar 

  14. Kumar G, Dasgupta I (2021) Variability, functions and interactions of plant virus movement proteins: what do we know so far? Microorganisms 9:695. https://doi.org/10.3390/microorganisms9040695

    Article  CAS  Google Scholar 

  15. Wu F-H, Shen S-C, Lee L-Y et al (2009) Tape-Arabidopsis sandwich – a simpler Arabidopsis protoplast isolation method. Plant Methods 5:16. https://doi.org/10.1186/1746-4811-5-16

    Article  CAS  Google Scholar 

  16. Priyadarshani SVGN, Hu B, Li W et al (2018) Simple protoplast isolation system for gene expression and protein interaction studies in pineapple (Ananas comosus L.). Plant Methods 14:95. https://doi.org/10.1186/s13007-018-0365-9

    Article  CAS  Google Scholar 

  17. Fontes EPB, Eagle PA, Sipe PS, Luckow VA, Hanley-Bowdoin L (1994) Interaction between a geminivirus replication protein and origin DNA is essential for viral replication. J Biol Chem 269:8459–8465. https://doi.org/10.1016/S0021-9258(17)37216-2

    Article  CAS  Google Scholar 

  18. Fontes EPB, Gladfelter HJ, Schaffer RL, Petty ITD, Hanley-Bowdoin L (1994) Geminivirus replication origins have a modular organization. Plant Cell 6:405–416. https://doi.org/10.1105/tpc.6.3.405

    Article  CAS  Google Scholar 

  19. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

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Acknowledgments

This work was partly supported by the Brazilian research funding agencies CNPq, Fapemig, and Capes (Print Program).

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Authors and Affiliations

  1. National Institute of Science and Technology in Plant-Pest Interactions, Bioagro, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil

    Laura Gonçalves Costa Martins & Elizabeth P. B. Fontes

  2. Departament of Biochemistry and Molecular Biology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil

    Elizabeth P. B. Fontes

Authors
  1. Laura Gonçalves Costa Martins
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  2. Elizabeth P. B. Fontes
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Editors and Affiliations

  1. Departamento de Bioquimica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil

    Elizabeth P.B. Fontes

  2. Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland

    Kristiina Mäkinen

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Martins, L.G.C., Fontes, E.P.B. (2024). Replication Assay of Begomovirus in Arabidopsis Protoplasts. In: Fontes, E.P., Mäkinen, K. (eds) Plant-Virus Interactions. Methods in Molecular Biology, vol 2724. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3485-1_9

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  • DOI: https://doi.org/10.1007/978-1-0716-3485-1_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3484-4

  • Online ISBN: 978-1-0716-3485-1

  • eBook Packages: Springer Protocols

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