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Catharanthus roseus pp 249–262Cite as

EASI Transformation Protocol: An Agrobacterium-Mediated Transient Transformation Protocol for Catharanthus roseus Seedlings

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2505))

Abstract

Catharanthus roseus produces medicinal terpenoid indole alkaloids, including the critical anti-cancer compounds vinblastine and vincristine in its leaves. Recently, we developed a highly efficient transient expression method relying on Agrobacterium-mediated transformation of seedlings to facilitate rapid and high-throughput studies on the regulation of terpenoid indole alkaloid biosynthesis in C. roseus . We detail our optimized protocol known as efficient Agrobacterium-mediated seedling infiltration method (EASI), including the development of constructs used in EASI and an example experimental design that includes appropriate controls. We applied our EASI method to rapidly screen and evaluate transcriptional activators and repressors and promoter activity. Our EASI method can be used for promoter transactivation studies or transgene overexpression paired with downstream analyses like quantitative PCR or metabolite analysis. Our protocol takes about 16 days from sowing seeds to obtaining the results of the experiment.

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References

  1. Sanford JC (1990) Biolistic plant transformation. Physiol Plant 79:206–209. https://doi.org/10.1111/j.1399-3054.1990.tb05888.x

    Article  CAS  Google Scholar 

  2. Torney F, Trewyn BG, Lin VS-Y, Wang K (2007) Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat Nanotechnol 2:295–300. https://doi.org/10.1038/nnano.2007.108

    Article  CAS  PubMed  Google Scholar 

  3. Yoo S-D, Cho Y-H, 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  PubMed  Google Scholar 

  4. Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y (2005) Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat Biotechnol 23:718–723. https://doi.org/10.1038/nbt1094

    Article  CAS  PubMed  Google Scholar 

  5. McBride KE, Summerfelt KR (1990) Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol Biol 14:269–276. https://doi.org/10.1007/BF00018567

    Article  CAS  PubMed  Google Scholar 

  6. Christou P, McCabe DE, Swain WF (1988) Stable transformation of soybean callus by DNA-coated gold particles. Plant Physiol 87:671–674. https://doi.org/10.1104/pp.87.3.671

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sidorov V, Gilbertson L, Addae P, Duncan D (2006) Agrobacterium-mediated transformation of seedling-derived maize callus. Plant Cell Rep 25:320–328. https://doi.org/10.1007/s00299-005-0058-5

    Article  CAS  PubMed  Google Scholar 

  8. Kapila J, De Rycke R, Van Montagu M, Angenon G (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–108. https://doi.org/10.1016/S0168-9452(96)04541-4

    Article  CAS  Google Scholar 

  9. Verweij W, Spelt C, Di Sansebastiano GP, Vermeer J, Reale L, Ferranti F, Koes R, Quattrocchio F (2008) An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour. Nat Cell Biol 10:1456–1462. https://doi.org/10.1038/ncb1805

    Article  CAS  PubMed  Google Scholar 

  10. Pan Q, Saiman MZ, Mustafa NR, Verpoorte R, Tang K (2016) A simple and rapid HPLC-DAD method for simultaneously monitoring the accumulation of alkaloids and precursors in different parts and different developmental stages of Catharanthus roseus plants. J Chromatogr B 1014:10–16. https://doi.org/10.1016/j.jchromb.2016.01.034

    Article  CAS  Google Scholar 

  11. Mortensen S, Bernal-Franco D, Cole LF, Sathitloetsakun S, Cram EJ, Lee-Parsons CWT (2019) EASI transformation: an efficient transient expression method for analyzing gene function in Catharanthus roseus seedlings. Front Plant Sci 10:1–17. https://doi.org/10.3389/fpls.2019.00755

    Article  Google Scholar 

  12. Young JM, Kuykendall LD, Martínez-Romero E, Kerr A, Sawada H (2001) A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi. Int J Syst Evol Microbiol 51:89–103. https://doi.org/10.1099/00207713-51-1-89

    Article  CAS  PubMed  Google Scholar 

  13. Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67:16–37, table of contents. https://doi.org/10.1128/MMBR.67.1.16

  14. Tsuda K, Qi Y, Nguyen LV, Bethke G, Tsuda Y, Glazebrook J, Katagiri F (2012) An efficient Agrobacterium-mediated transient transformation of Arabidopsis. Plant J 69:713–719. https://doi.org/10.1111/j.1365-313X.2011.04819.x

    Article  CAS  PubMed  Google Scholar 

  15. Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025. https://doi.org/10.1038/nprot.2006.286

    Article  CAS  PubMed  Google Scholar 

  16. Wirz H, Sauer-budge AF, Briggs J, Sharpe A, Shu S, Sharon A (2012) Automated production of plant-based vaccines and pharmaceuticals. J Lab Autom 17:449–457. https://doi.org/10.1177/2211068212460037

    Article  PubMed  Google Scholar 

  17. Pogue GP, Vojdani F, Palmer KE, Hiatt E, Hume S, Phelps J, Long L, Bohorova N, Kim D, Pauly M, Velasco J, Whaley K, Zeitlin L, Garger SJ, White E, Bai Y, Haydon H, Bratcher B (2010) Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems. Plant Biotechnol J 8:638–654. https://doi.org/10.1111/j.1467-7652.2009.00495.x

    Article  CAS  PubMed  Google Scholar 

  18. An G, Watson BD, Chiang CC (1986) Transformation of tobacco, tomato, potato, and Arabidopsis thaliana using a binary Ti vector system. Plant Physiol 81:301–305. https://doi.org/10.1104/pp.81.1.301

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Simmons CW, Vandergheynst JS, Upadhyaya SK (2009) A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression. Biotechnol Bioeng 102:965–970. https://doi.org/10.1002/bit.22118

    Article  CAS  PubMed  Google Scholar 

  20. Mortensen S, Weaver JD, Sathitloetsakun S, Cole LF, Rizvi NF, Cram EJ, Lee-Parsons CWT (2019) The regulation of ZCT1, a transcriptional repressor of monoterpenoid indole alkaloid biosynthetic genes in Catharanthus roseus. Plant Direct 3:1–13. https://doi.org/10.1002/pld3.193

    Article  Google Scholar 

  21. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One 6. https://doi.org/10.1371/journal.pone.0016765

  22. Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S (2012) Fast track assembly of multigene constructs using golden gate cloning and the MoClo system. Bioeng Bugs 3:38–43. https://doi.org/10.4161/bbug.3.1.18223

    Article  PubMed  Google Scholar 

  23. Pazour GJ, Ta CN, Das A (1992) Constitutive mutations of Agrobacterium tumefaciens transcriptional activator virG. J Bacteriol 174:4169–4174. https://doi.org/10.1128/jb.174.12.4169-4174.1992

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Schweizer F, Colinas M, Pollier J, Van Moerkercke A, Vanden Bossche R, de Clercq R, Goossens A (2018) An engineered combinatorial module of transcription factors boosts production of monoterpenoid indole alkaloids in Catharanthus roseus. Metab Eng 48:150–162. https://doi.org/10.1016/j.ymben.2018.05.016

    Article  CAS  PubMed  Google Scholar 

  25. Weaver J, Goklany S, Rizvi N, Cram EJ, Lee-Parsons CWT (2014) Optimizing the transient Fast Agro-mediated Seedling Transformation (FAST) method in Catharanthus roseus seedlings. Plant Cell Rep 33:89–97. https://doi.org/10.1007/s00299-013-1514-2

    Article  CAS  PubMed  Google Scholar 

  26. Chetty VJ, Ceballos N, Garcia D, Narváez-Vásquez J, Lopez W, Orozco-Cárdenas ML (2013) Evaluation of four Agrobacterium tumefaciens strains for the genetic transformation of tomato (Solanum lycopersicum L.) cultivar Micro-Tom. Plant Cell Rep 32:239–247. https://doi.org/10.1007/s00299-012-1358-1

    Article  CAS  PubMed  Google Scholar 

  27. Marillonnet S, Grützner R (2020) Synthetic DNA assembly using golden gate cloning and the hierarchical modular cloning pipeline. Curr Protoc Mol Biol 130:1–33. https://doi.org/10.1002/cpmb.115

    Article  CAS  Google Scholar 

  28. Engler C, Youles M, Gruetzner R, Ehnert TM, Werner S, Jones JDG, Patron NJ, Marillonnet S (2014) A Golden Gate modular cloning toolbox for plants. ACS Synth Biol 3:839–843. https://doi.org/10.1021/sb4001504

    Article  CAS  PubMed  Google Scholar 

  29. Gantner J, Ordon J, Ilse T, Kretschmer C, Gruetzner R, Löfke C, Dagdas Y, Bürstenbinder K, Marillonnet S, Stuttmann J (2018) Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system. PLoS One 13. https://doi.org/10.1371/journal.pone.0197185

  30. Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet MGG 204:383–396. https://doi.org/10.1007/BF00331014

    Article  CAS  Google Scholar 

  31. Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629. https://doi.org/10.1038/318624a0

    Article  Google Scholar 

  32. Natesan S, Rivera VM, Molinari E, Gilman M (1997) Transcriptional squelching re-examined. Nature 390:349–350. https://doi.org/10.1038/37019

    Article  CAS  PubMed  Google Scholar 

  33. Fullner KJ, Nester EW (1996) Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J Bacteriol 178:1498–1504. https://doi.org/10.1128/jb.178.6.1498-1504.1996

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sung Y-C, Lin C-P, Chen J-C (2014) Optimization of virus-induced gene silencing in Catharanthus roseus. Plant Pathol 63:1159–1167. https://doi.org/10.1111/ppa.12186

    Article  CAS  Google Scholar 

  35. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907. https://doi.org/10.1002/j.1460-2075.1987.tb02730.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Li JF, Park E, Von Arnim AG, Nebenführ A (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5:6. https://doi.org/10.1186/1746-4811-5-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Mortensen S (2019) Save time with transient plant leaf transformations. https://blog.addgene.org/save-time-with-transient-plant-leaf-transformations. Accessed 27 Mar 2021

  38. Applied Biosystems (2011) SYBR® Green PCR Master Mix and SYBR® Green RT-PCR Reagents Kit : User Guide

    Google Scholar 

  39. GeneCopoeia (2017) Luc-Pair™ Duo-Luciferase Assay Kit 2.0 : User Manual

    Google Scholar 

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Acknowledgment

This work was supported by the National Science Foundation (NSF) MCB Award #1516371 and #2031237 to CL-P and EC.

Author information

Author notes

  1. Samuel Mortensen and Lauren F. Cole contributed equally.

Authors and Affiliations

  1. Department of Biology, Northeastern University, Boston, MA, USA

    Samuel Mortensen, Diana Bernal-Franco, Suphinya Sathitloetsakun & Erin J. Cram

  2. Department of Bioengineering, Northeastern University, Boston, MA, USA

    Lauren F. Cole

  3. Department of Chemical Engineering, Northeastern University, Boston, MA, USA

    Diana Bernal-Franco & Carolyn W. T. Lee-Parsons

  4. Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA, USA

    Suphinya Sathitloetsakun & Carolyn W. T. Lee-Parsons

Authors
  1. Samuel Mortensen
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  2. Lauren F. Cole
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  3. Diana Bernal-Franco
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  4. Suphinya Sathitloetsakun
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  5. Erin J. Cram
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  6. Carolyn W. T. Lee-Parsons
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Corresponding author

Correspondence to Carolyn W. T. Lee-Parsons .

Editor information

Editors and Affiliations

  1. EA2106 Biomolécules et Biotechnologies Végétales, Université de Tours, Tours, France

    Vincent Courdavault

  2. EA2106 Biomolécules et Biotechnologies Végétales, Université de Tours, Tours, France

    Sebastien Besseau

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Mortensen, S., Cole, L.F., Bernal-Franco, D., Sathitloetsakun, S., Cram, E.J., Lee-Parsons, C.W.T. (2022). EASI Transformation Protocol: An Agrobacterium-Mediated Transient Transformation Protocol for Catharanthus roseus Seedlings. In: Courdavault, V., Besseau, S. (eds) Catharanthus roseus. Methods in Molecular Biology, vol 2505. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2349-7_18

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  • DOI: https://doi.org/10.1007/978-1-0716-2349-7_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2348-0

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